Human cytomegalovirus neutralizing antibodies and use thereof

ABSTRACT

The invention relates to neutralizing antibodies, and antibody fragments thereof, having high potency in neutralizing hCMV, wherein said antibodies and antibody fragments are specific for one, or a combination of two or more, hCMV gene UL products. The invention also relates to immortalized B cells that produce, and to epitopes that bind to, such antibodies and antibody fragments. In addition, the invention relates to the use of the antibodies, antibody fragments, and epitopes in screening methods as well as in the diagnosis, prevention, and therapy of disease.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 15/156,786 (allowed),filed on May 17, 2016 which is a divisional of U.S. Pat. No. 9,371,372,issued on Jun. 21, 2016, which is a divisional of U.S. Pat. No.9,249,213, issued on Feb. 2, 2016, which is a divisional of U.S. Pat.No. 9,127,049, issued on Sep. 8, 2015, which is a divisional of U.S.Pat. No. 8,765,132, issued on Jul. 1, 2014, which is a divisional ofU.S. Pat. No. 8,435,524, issued on May 7, 2013, which is a divisional ofU.S. Pat. No. 8,287,870, issued on Oct. 16, 2012, which is a divisionalof U.S. Pat. No. 8,124,093, issued on Feb. 28, 2012, which claimspriority to U.S. Provisional Application No. 61/081,334, filed on Jul.16, 2008, the contents of which are hereby incorporated by reference.

BACKGROUND

Human cytomegalovirus (hCMV) is a widely distributed pathogen that maycause severe pathology in immunosuppressed adults and upon infection ofthe fetus and has been implicated in chronic diseases such asatherosclerosis. hCMV infects multiple cell types including fibroblasts,endothelial, epithelial and hematopoietic cells [1]. In vitro propagatedattenuated strains of hCMV, which are being developed as candidatevaccines, have lost the tropism for endothelial cells, while retainingthe capacity to infect fibroblasts [2]. Two viral glycoprotein complexesare believed to control the cellular tropism of hCMV. A complex ofglycoproteins such as gH, gL and gO appears to be required for infectionof fibroblasts, while a complex of gH, gL and proteins encoded by theUL131-UL128 genes is implicated in infection of endothelial cells,epithelial cells and dendritic cells [2-8].

Hyperimmune globulins are already commercialized for the prophylaxis ofhCMV disease associated with transplantation and recent evidenceindicates that they have therapeutic effect in pregnant women [9]. Thistherapeutic approach is limited by the low amount of neutralizingantibody that can be transferred and for this reason the availability ofhuman antibodies (such as human monoclonal antibodies) with highneutralizing capacity would be highly desirable. Although someantibodies to gH, gB and UL128 and UL130 gene products have demonstratedin vitro neutralizing activities [7, 10, 11] and an antibody to gH wasevaluated in clinical trials (that were discontinued due to lack oftherapeutic effects), the neutralizing potency of the antibodiesisolated so far is modest. Neutralization by these antibodies wasobserved at antibody concentrations ranging from 0.5 to 20 μg/ml.Further, the current methods typically measure the neutralizing potencyof anti-hCMV antibodies using fibroblasts as target cells. However, hCMVis also known to cause pathology by infecting other cell types such asendothelial, epithelial cells and leukocytes. Known antibodies to UL128and UL130 show very low potency in neutralizing infection of endothelialcells [7] and there do not appear to be any monoclonal antibodiesavailable that would be capable of neutralizing infection ofnon-fibroblast target cells with high potency.

There is therefore a need for antibodies that neutralize hCMV infection,particularly hCMV infection of non-fibroblast target cells, with highpotency, as well as the elucidation of the target(s) to which suchantibodies bind.

SUMMARY OF INVENTION

The invention is based, in part, on the discovery of novel antibodiesthat neutralize hCMV infection with high potency as well as novelepitopes to which the antibodies of the invention bind. Accordingly, inone aspect, the invention comprises an antibody and antigen bindingfragments thereof that have high potency in neutralizing hCMV.

In one embodiment of the invention, the invention comprises a monoclonalantibody, or an antigen binding fragment thereof, that binds to anepitope in the hCMV UL128 protein, wherein the antibody neutralizes hCMVinfection. In another embodiment of the invention, the inventioncomprises an antibody, or an antigen binding fragment thereof, thatbinds to an epitope formed by the hCMV proteins gH, gL, UL128 and UL130,the hCMV proteins UL128, UL130 and UL131A, or the hCMV proteins UL130and UL131A, wherein the antibody neutralizes hCMV infection.

In yet another embodiment of the invention, the invention comprises anantibody, or an antigen binding fragment thereof, comprising at leastone complementarity determining region (“CDR”) sequence having at least95% sequence identity to any one of SEQ ID NOs: 188-193, 204, 205, 210,174-177, 149, 178, 65-70, 81-86, 97-102, 129-134, 145-150, 113, 161-164,1-6, 17-22, 33-38, 49-54, or 114-118, wherein the antibody neutralizeshCMV infection.

In yet another embodiment of the invention, the invention comprises aheavy chain CDR1 selected from the group consisting of SEQ ID NOs: 188,174, 65, 81, 97, 129, 145, 113, 1, 17, 33, and 49; a heavy chain CDR2selected from the group consisting of SEQ ID NOs: 189, 204, 175, 66, 82,98, 130, 146, 161, 2, 2, 18, 34, 50, and 114; and a heavy chain CDR3selected from the group consisting of SEQ ID NOs: 190, 205, 210, 176,67, 83, 99, 131, 147, 162, 3, 19, 35, 51, and 115, wherein the antibodyneutralizes hCMV infection. In yet another embodiment of the invention,the invention comprises an antibody, or an antigen binding fragmentthereof, comprising a light chain CDR1 selected from the groupconsisting of SEQ ID NOs: 191, 177, 68, 84, 100, 132, 148, 163, 4, 20,36, 52, and 116; a light chain CDR2 selected from the group consistingof SEQ ID NOs: 192, 149, 69, 85, 101, 133, 5, 21, 37, 53, and 117; and alight chain CDR3 selected from the group consisting of SEQ ID NOs: 193,178, 70, 86, 102, 134, 150, 164, 6, 22, 38, 54, and 118, wherein theantibody neutralizes hCMV infection.

In still another embodiment of the invention, the invention comprises anantibody, or an antigen binding fragment thereof, wherein the antibodycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 200 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 201; or a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 200 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:213; or a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 208 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 201; or a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 208 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 213; ora heavy chain variable region comprising the amino acid sequence of SEQID NO: 212 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 201; or a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 212 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 213; or a heavychain variable region comprising the amino acid sequence of SEQ ID NO:184 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 185; or a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 77 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 78; or a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 93 anda light chain variable region comprising the amino acid sequence of SEQID NO: 94; or a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 109 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 110; or a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 141 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:142; or a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 157 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 158; or a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 170 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 171; ora heavy chain variable region comprising the amino acid sequence of SEQID NO: 13 and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 14; or a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 29 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 30; or a heavychain variable region comprising the amino acid sequence of SEQ ID NO:45 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 46; or a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 61 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 62; or a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 125 anda light chain variable region comprising the amino acid sequence of SEQID NO: 126, and wherein the antibody neutralizes hCMV infection.

In a further embodiment of the invention, the invention comprises anantibody, or an antigen binding fragment thereof, that neutralizesinfection of endothelial cells, epithelial cells, retinal cells, myeloidcells, dendritic cells, fibroblasts, or mesenchymal stromal cells by aclinical isolate of hCMV, wherein the concentration of antibody requiredfor 90% neutralisation of hCMV is 1.2 μg/ml or less. In anotherembodiment of the invention, the invention comprises an antibody, or anantigen binding fragment thereof, that neutralizes infection ofendothelial cells, epithelial cells, retinal cells, myeloid cells,dendritic cells, fibroblasts, or mesenchymal stromal cells by a clinicalisolate of hCMV, wherein the concentration of antibody required for 90%neutralisation of hCMV is 10 μg/ml or less, and wherein the antibody isnot MSL-109 or 8F9.

In yet another embodiment of the invention, the invention comprises anantibody, or an antigen binding fragment thereof, comprising at leastone CDR sequence having at least 95% sequence identity to any one of SEQID NOs: 216-221, 232-235, 149, 236, 246-251, 278-283, 296-301, 312,316-321, 332, 336-341, 352, 360, 361 or 262-267, wherein the antibodyneutralizes hCMV infection.

In yet another embodiment of the invention, the invention comprises anantibody, or an antigen binding fragment thereof, comprising a heavychain CDR1 selected from the group consisting of SEQ ID NOs: 216, 232,246, 278, 296, 316, 336, 352, 360 and 262; a heavy chain CDR2 selectedfrom the group consisting of SEQ ID NOs: 217, 233, 247, 279, 297, 312,317, 337 and 263; and a heavy chain CDR3 selected from the groupconsisting of SEQ ID NOs: 218, 234, 248, 280, 298, 318, 332, 338, and264, wherein the antibody neutralizes hCMV infection.

In yet another embodiment of the invention, the invention comprises anantibody, or an antigen binding fragment thereof, comprising a lightchain CDR1 selected from the group consisting of SEQ ID NOs: 219, 235,249, 281, 299, 319, 339 and 265; a light chain CDR2 selected from thegroup consisting of SEQ ID NOs: 220, 149, 250, 282, 300, 320, 340 and266; and a light chain CDR3 selected from the group consisting of SEQ IDNOs: 221, 236, 251, 283, 301, 321, 341, 361 and 267, wherein theantibody neutralizes hCMV infection.

In still another embodiment of the invention, the invention comprises anantibody, or an antigen binding fragment thereof, wherein the antibodycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 228 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 229; or a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 242 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:243; or a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 258 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 259; or a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 290, and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 291; ora heavy chain variable region comprising the amino acid sequence of SEQID NO: 294, and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 291; or a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 308, and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 309; or a heavychain variable region comprising the amino acid sequence of SEQ ID NO:314, and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 309; or a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 328, and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 329; or a heavychain variable region comprising the amino acid sequence of SEQ ID NO:334, and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 329; or a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 348 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 349; or a heavychain variable region comprising the amino acid sequence of SEQ ID NO:357 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 291; or a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 367 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 368; or a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 274 anda light chain variable region comprising the amino acid sequence of SEQID NO: 275, and wherein the antibody neutralizes hCMV infection.

The invention further comprises an antibody, or an antigen bindingfragment thereof, produced by immortalised B cell clone 8I21, 2C12,8C15, 4N10, 11B12, 3G16, 4H9, 6B4, 10C6, or 6L3 deposited with theAdvanced Biotechnology Center (ABC), Largo Rossana Benzi 10, 16132 Genoa(Italy), under the terms of the Budapest Treaty, on Jul. 9, 2008 (underAccession Numbers PD 08005, PD 08007, PD 08006, PD 08009, PD 08011, PD08012, PD 08013, PD 08004, PD 08014, and PD 08010, respectively) and byimmortalized B cell clone 7H3 deposited on Jul. 16, 2008 under AccessionNumber PD 08017. Antibodies and antigen binding fragments thereof, withthe same amino acid sequence as those expressed from the aforementioneddeposited immortalised B cells are also considered to be within thescope of the invention.

In another aspect, the invention comprises a nucleic acid moleculecomprising a polynucleotide encoding an antibody or antibody fragment ofthe invention that neutralizes hCMV infection. In yet another aspect,the invention comprises a cell expressing an antibody of the invention.In still another aspect, the invention comprises an isolated or purifiedimmunogenic polypeptide comprising an epitope that binds to an antibodyof the invention.

The invention further comprises a pharmaceutical composition comprisingan antibody of the invention or an antigen binding fragment thereof, anucleic acid molecule of the invention, or an immunogenic polypeptide ofthe invention, and a pharmaceutically acceptable diluent or carrier. Theinvention also comprises a pharmaceutical composition comprising a firstantibody or an antigen binding fragment thereof, and a second antibody,or an antigen binding fragment thereof, wherein the first antibody is anantibody of the invention, and the second antibody is an antibody thatneutralizes hCMV infection.

Use of an antibody of the invention, or an antigen binding fragmentthereof, a nucleic acid of the invention, an immunogenic polypeptide ofthe invention, or a pharmaceutical composition of the invention (i) inthe manufacture of a medicament for the treatment of hCMV infection,(ii) in a vaccine, or (iii) in diagnosis of hCMV infection is alsocontemplated to be within the scope of the invention. Further, use of anantibody of the invention, or an antigen binding fragment thereof, formonitoring the quality of anti-hCMV vaccines by checking that theantigen of said vaccine contains the specific epitope in the correctconformation is also contemplated to be within the scope of theinvention.

In a further aspect, the invention comprises an epitope whichspecifically binds to an antibody of any one of the invention, or anantigen binding fragment thereof, for use (i) in therapy, (ii) in themanufacture of a medicament for treating hCMV infection, (iii) as avaccine, or (iv) in screening for ligands able to neutralise hCMVinfection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows staining of HEK293T cells transfected with hCMV UL128,UL130, UL131A, gH and gL genes, alone or in different combinations, byrepresentative monoclonal antibodies (15D8, 2C12 and 8I21).

FIG. 2 shows cross-competition experiments in which HEK293T cellstransfected with hCMV gH (A) or gB (B) gene were first incubated with anunlabeled competitor antibody followed by staining with a biotinylatedanti-gH or anti-gB antibody.

FIG. 3 shows staining of HEK293T cells expressing either the wild typeVR1814 UL128 gene or a pan-mutated UL128 gene by human monoclonalantibody 15D8 and a non-competing anti-UL128 mouse monoclonal antibody.The pan-mutated UL128 gene contains substitutions of the wild typeVR1814 sequence with known variants described in other clinical isolatesand laboratory strains of hCMV.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in part, on the discovery of novel antibodiesthat neutralize hCMV infection with high potency as well as novelepitopes to which the antibodies of the invention bind. Such antibodiesare desirable, as only low concentrations are required in order toneutralize a given amount of virus. This facilitates higher levels ofprotection whilst administering lower amounts of antibody. Accordingly,in one aspect, the invention comprises a neutralizing antibody andantigen binding fragments thereof having high potency in neutralizinghCMV infection. Human monoclonal antibodies and the immortalised B cellclones that secrete such antibodies are also included within the scopeof the invention.

As used herein, the terms “fragment,” “antigen binding fragment” and“antibody fragment” are used interchangeably to refer to any fragment ofan antibody of the invention that retains the antigen-binding activityof the antibodies. Exemplary antibody fragments include, but are notlimited to, a single chain antibody, Fab, Fab′, F(ab′)2, Fv or scFv.

As used herein, the term “high potency” is used to refer to an antibodyof the invention or an antigen binding fragment thereof that neutralizeshCMV infection with an 1C₉₀ of less than about 2 μg/ml, (i.e. theconcentration of antibody required for 90% neutralisation of a clinicalisolate of hCMV is about 2 μg/ml or less, for example 1.9, 1.8, 1.75,1.7, 1.6, 1.5, 1.4, 1.3, 1.25, 1.2, 1.15, 1.1, or 1.05 μg/ml or less).In one embodiment, the antibody of the present invention, or antigenbinding fragment thereof, has an IC₉₀ of 1 μg/ml or less (i.e. 0.95,0.9, 0.85, 0.8, 0.75, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01μg/ml or less). In another embodiment, the antibody of the presentinvention, or antigen binding fragment thereof, has an IC₉₀ of 0.16μg/ml or less (i.e. 0.15, 0.125, 0.1, 0.075, 0.05, 0.025, 0.02, 0.015,0.0125, 0.01, 0.0075, 0.005, 0.004, 0.003, 0.002 μg/ml or less). Inanother embodiment, the antibody can neutralize hCMV infection at aconcentration of 0.016 μg/ml or less (i.e. at 0.015, 0.013, 0.01, 0.008,0.005, 0.003, 0.002, 0.001, 0.0005 μg/ml or less). This means that onlyvery low concentrations of antibody are required for 90% neutralisationof a clinical isolate of hCMV in vitro compared to the concentration ofknown antibodies, e.g., MSL-109, 8F9 or 3E3, required for neutralisationof the same titre of hCMV. Potency can be measured using a standardneutralisation assay as known to one of skill in the art.

In one embodiment, the invention provides an antibody, for example, amonoclonal antibody or a human monoclonal antibody, or an antigenbinding fragment thereof, that binds to an epitope in the hCMV UL128protein and neutralizes hCMV infection with an IC₉₀ of less than about 2μg/ml, for example 1.9, 1.8, 1.75, 1.7, 1.6, 1.5, 1.4, 1.3, 1.25, 1.2,1.15, 1.1, 1.05, 1, 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.6, 0.5, 0.4, 0.3,0.2, 0.15, 0.125, 0.1, 0.075, 0.05, 0.025, 0.02, 0.015, 0.0125, 0.01,0.0075, 0.005, 0.004, 0.003, 0.002 0.001, 0.0005 μg/ml or less.

In another embodiment, the invention provides an antibody, or an antigenbinding fragment thereof, that binds to an epitope formed by the hCMVproteins gH, gL, UL128 and UL130, and neutralizes hCMV infection with anIC₉₀ of less than about 2 μg/ml, for example 1.9, 1.8, 1.75, 1.7, 1.6,1.5, 1.4, 1.3, 1.25, 1.2, 1.15, 1.1, 1.05, 1, 0.95, 0.9, 0.85, 0.8,0.75, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.15, 0.125, 0.1, 0.075, 0.05,0.025, 0.02, 0.015, 0.0125, 0.01, 0.0075, 0.005, 0.004, 0.003, 0.0020.001, 0.0005 μg/ml or less.

In another embodiment, the invention provides an antibody, or an antigenbinding fragment thereof, that binds to an epitope formed by the hCMVproteins UL128, UL130, and UL131A, and neutralizes hCMV infection withan IC₉₀ of less than about 2 μg/ml, for example 1.9, 1.8, 1.75, 1.7,1.6, 1.5, 1.4, 1.3, 1.25, 1.2, 1.15, 1.1, 1.05, 1, 0.95, 0.9, 0.85, 0.8,0.75, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.15, 0.125, 0.1, 0.075, 0.05,0.025, 0.02, 0.015, 0.0125, 0.01, 0.0075, 0.005, 0.004, 0.003, 0.0020.001, 0.0005 μg/ml or less.

In another embodiment, the invention provides an antibody, or an antigenbinding fragment thereof, that binds to an epitope formed by the hCMVproteins UL130 and UL131A, and neutralizes hCMV infection with an IC₉₀of less than about 2 μg/ml, for example 1.9, 1.8, 1.75, 1.7, 1.6, 1.5,1.4, 1.3, 1.25, 1.2, 1.15, 1.1, 1.05, 1, 0.95, 0.9, 0.85, 0.8, 0.75,0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.15, 0.125, 0.1, 0.075, 0.05, 0.025,0.02, 0.015, 0.0125, 0.01, 0.0075, 0.005, 0.004, 0.003, 0.002 0.001,0.0005 μg/ml or less.

In yet another embodiment, the invention provides an antibody, or anantigen binding fragment thereof, that binds to an epitope in the hCMVgH protein and neutralizes hCMV infection with an IC₉₀ of less thanabout 2 μg/ml, for example 1.9, 1.8, 1.75, 1.7, 1.6, 1.5, 1.4, 1.3,1.25, 1.2, 1.15, 1.1, 1.05, 1, 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.6,0.5, 0.4, 0.3, 0.2, 0.15, 0.125, 0.1, 0.075, 0.05, 0.025, 0.02, 0.015,0.0125, 0.01, 0.0075, 0.005, 0.004, 0.003, 0.002 0.001, 0.0005 μg/ml orless.

In yet another embodiment, the invention provides an antibody, or anantigen binding fragment thereof, that binds to an epitope in the hCMVgB protein and neutralizes hCMV infection with an IC₉₀ of less thanabout 2 μg/ml, for example 1.9, 1.8, 1.75, 1.7, 1.6, 1.5, 1.4, 1.3,1.25, 1.2, 1.15, 1.1, 1.05, 1, 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.6,0.5, 0.4, 0.3, 0.2, 0.15, 0.125, 0.1, 0.075, 0.05, 0.025, 0.02, 0.015,0.0125, 0.01, 0.0075, 0.005, 0.004, 0.003, 0.002 0.001, 0.0005 μg/ml orless.

In another embodiment, the invention provides an antibody, or an antigenbinding fragment thereof, that binds to an epitope formed by the hCMVproteins gM and gN and neutralizes hCMV infection with an IC₉₀ of lessthan about 2 μg/ml, for example 1.9, 1.8, 1.75, 1.7, 1.6, 1.5, 1.4, 1.3,1.25, 1.2, 1.15, 1.1, 1.05, 1, 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.6,0.5, 0.4, 0.3, 0.2, 0.15, 0.125, 0.1, 0.075, 0.05, 0.025, 0.02, 0.015,0.0125, 0.01, 0.0075, 0.005, 0.004, 0.003, 0.002 0.001, 0.0005 μg/ml orless.

Antibodies of the Invention

The invention provides antibodies having particularly high potency inneutralizing hCMV. As used herein, an “antibody that neutralizes” is onethat prevents, reduces, delays or interferes with the ability of apathogen, e.g., hCMV, to initiate and/or perpetuate an infection in ahost. The antibodies of the invention and antigen-binding fragmentsthereof are able to neutralize hCMV infection of several kinds of cells.In one embodiment, an antibody according to the invention neutralizesinfection of epithelial cells, retinal cells, endothelial cells, myeloidcells and dendritic cells. The antibodies of the invention may alsoneutralize hCMV infection of fibroblasts and mesenchymal stromal cells.These antibodies can be used as prophylactic or therapeutic agents uponappropriate formulation, or as a diagnostic tool, as described herein.

The antibodies of the invention may be monoclonal antibodies, humanantibodies, or recombinant antibodies. In one embodiment, the antibodiesof the invention are monoclonal antibodies, e.g., human monoclonalantibodies. The invention also provides fragments of the antibodies ofthe invention, particularly fragments that retain the antigen-bindingactivity of the antibodies and neutralize hCMV infection. Although thespecification, including the claims, may, in some places, referexplicitly to antibody fragment(s), variant(s) and/or derivative(s) ofantibodies, it is understood that the term “antibody” or “antibody ofthe invention” includes all categories of antibodies, namely, antibodyfragment(s), variant(s) and derivative(s) of antibodies.

In one embodiment, the antibodies of the invention and antigen bindingfragments thereof bind to one or more hCMV proteins. The antibodies ofthe invention may bind to an epitope formed by a single hCMV protein orby a combination of two or more hCMV proteins. Exemplary hCMV proteinsinclude, but are not limited to, products of viral genes UL55 (envelopeglycoprotein B, “gB”), UL75 (envelope glycoprotein H, “gH”), UL100(glycoprotein M, “gM”), UL73 (glycoprotein N, “gN”), UL115 (glycoproteinL, “gL”), UL74 (glycoprotein O, “gO”), UL128 (glycoprotein UL128,“UL128”), UL130 (glycoprotein UL130, “UL130”) or UL131A (glycoproteinUL131A, “UL131A”). In one embodiment, the antibodies of the inventionbind to an epitope formed by a single hCMV protein. In anotherembodiment, the antibodies bind to an epitope formed by the combinationof 2, 3, or more hCMV proteins.

In an exemplary embodiment, the invention comprises an antibody, or anantibody fragment thereof, that binds to an epitope in the hCMV proteinUL128, or to an epitope formed by the hCMV proteins UL130 and UL131A, orto an epitope formed by the hCMV proteins UL128, UL130 and UL131A, or toan epitope formed by the hCMV proteins gH, gL, UL128, and UL130, or toan epitope in the hCMV protein gH, or the hCMV protein gB or to anepitope formed by the hCMV proteins gM and gN.

In one embodiment, the invention comprises an antibody, or an antibodyfragment thereof, that binds to an epitope in UL128. In anotherembodiment, the invention comprises an antibody, or an antibody fragmentthereof, that binds to an epitope formed by UL130 and UL131A. As usedherein, an epitope formed by UL130 and UL131A means that the epitope maybe formed by both UL130 and UL131A protein or may be formed by one ofthe two proteins, the presence of the other protein being necessary forantibody binding. In yet another embodiment, the invention comprises anantibody, or an antibody fragment thereof, that binds to an epitopeformed by UL128, UL130 and UL131A. As used herein, an epitope formed byUL128, UL130 and UL131A means that the epitope may be formed by allthree proteins (UL128, UL130 and UL131A) or may be formed by one or moreprotein(s), the presence of the other protein(s) being necessary forantibody binding. In still another embodiment, the invention comprisesan antibody, or an antibody fragment thereof, that binds to an epitopeformed by gH, gL, UL128, and UL130. As used herein, an epitope formed bygH, gL, UL128, and UL130 means that the epitope may be formed by allfour proteins (gH, gL, UL128, and UL130) or may be formed by one or moreof the four protein(s), the presence of the other protein(s) beingnecessary for antibody binding. In another embodiment, the inventioncomprises an antibody, or an antibody fragment thereof, that binds to anepitope formed by gM and gN. As used herein, an epitope formed by gM andgN means that the epitope may be formed by both gM and gN or may beformed by one of the two proteins, the presence of the other proteinbeing necessary for antibody binding.

The sequences of the heavy chains and light chains of several exemplaryantibodies of the invention, each comprising three CDRs on the heavychain and three CDRs on the light chain have been determined. Theposition of the CDR amino acids are defined according to the IMGTnumbering system [12, 13, 14]. The sequences of the CDRs, heavy chains,light chains as well as the sequences of the nucleic acid moleculesencoding the CDRs, heavy chains, light chains are disclosed in thesequence listing. Table 1 provides the SEQ ID NOs. for the sequences ofthe six CDRs of the exemplary antibodies of the invention. Tables 2 and3 provide the SEQ ID NOs for the sequences of the heavy and lightchains, respectively, of the exemplary antibodies of the invention, andTable 4 provides the SEQ ID NOs for the sequences of the nucleic acidmolecules encoding the CDRs, heavy chains and light chains of theantibodies.

TABLE 1 SEQ ID NOs. for SEQ ID NOs. for CDRH1, CDRH3, CDRL1, CDRL2,Antibody CDRH3 CDRL3 15D8 188, 189, 190 191, 192, 193 15D8 variant 1188, 204, 205 191, 192, 193 15D8 variant 2 188, 189, 210 191, 192, 1934N10 1, 2, 3 4, 5, 6 10F7 17, 18, 19 20, 21, 22 10P3 33, 34, 35 36, 37,38 4I22 49, 50, 51 52, 53, 54 8L13 113, 114, 115 116, 117, 118 2C12 65,66, 67 68, 69, 70 8C15 81, 82, 83 84, 85, 86 9I6 97, 98, 99 100, 101,102 7B13 129, 130, 131 132, 133, 134 8J16 145, 146, 147 148, 149, 1508I21 174, 175, 176 177, 149, 178 7I13 113, 161, 162 163, 149, 164 7H3316, 317, 318 319, 320, 321 7H3 variant 1 316, 317, 332 319, 320, 3216B4 336, 337, 338 339, 340, 341 5F1 278, 279, 280 281, 282, 283 10C6352, 279, 280 281, 282, 283 4H9 296, 297, 298 299, 300, 301 4H9 variant1 296, 312, 298 299, 300, 301 11B12 232, 233, 234 235, 149, 236 13H11216, 217, 218 219, 220, 221 3G16 246, 247, 248 249, 250, 251 2B11 360,279, 280 281, 282, 361 6L3 262, 263, 264 265, 266, 267

TABLE 2 Antibody SEQ ID NOs for Heavy Chains 15D8 200 15D8 variant 1 20815D8 variant 2 212 4N10 13 10F7 29 10P3 45 4I22 61 8L13 125 2C12 77 8C1593 9I6 109 7B13 141 8J16 157 8I21 184 7I13 170 7H3 328 7H3 variant 1 3346B4 348 5F1 290 5F1 variant 1 294 10C6 357 4H9 308 4H9 variant 1 31411B12 242 13H11 228 3G16 258 2B11 367 6L3 274

TABLE 3 Antibody SEQ ID NO for Light Chains 15D8 201 15D8 variant 1 20115D8 variant 2 213 4N10 14 10F7 30 10P3 46 4I22 62 8L13 126 2C12 78 8C1594 9I6 110 7B13 142 8J16 158 8I21 185 7I13 171 7H3 329 7H3 variant 1 3296B4 349 5F1 291 5F1 variant 1 291 10C6 291 4H9 309 4H9 variant 1 30911B12 243 13H11 229 3G16 259 2B11 368 6L3 275

TABLE 4 SEQ ID NO for Nucleic Acids encoding CDRs, Heavy Chains, LightChains and Variants (CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3 andvariants; Heavy Chain and variants; and Light Antibody Chains andvariants) 15D8 194-199 and 206, 207, 211; 202 and 209, 214; 203 and 2154N10 7-12; 15; 16 10F7 23-28; 31; 32 10P3 39-44; 47; 48 4I22 55-60; 63;64 8L13 119-124; 127; 128 2C12 71-76; 79; 80 8C15 87-92; 95; 96 9I6103-108, 111, 112 7B13 135-140; 143; 144 8J16 151-156; 159; 160 8I21179-182, 155, 183; 186; 187 7I13 165, 166, 167, 168, 155, 169; 172; 1737H3 322-327 and 333; 330 and 335; 331 6B4 342-347; 350; 351 5F1 284-289;292 and 295; 293 10C6 353-355, 287, 288, 356; 358; 359 4H9 302-307 and313; 310 and 315; 311 11B12 237-240, 155, 241; 244; 245 13H11 222-227;230; 231 3G16 252-257; 260; 261 2B11 362-364; 287, 365, 366; 369; 3706L3 268-273; 276; 277

In one embodiment, the antibodies or antibody fragments of the inventioncomprise one or more heavy or light chain CDRs of the exemplaryantibodies of the invention. In an exemplary embodiment, the antibodiesor antibody fragments of the invention comprise an amino acid sequenceselected from the group consisting of SEQ ID NOs: 188-193, 204-205, 210,1-6, 17-22, 33-38, 49-54, 113-118, 65-70, 81-86, 97-102, 129-134,145-150, 174-178, and 161-164.

In another embodiment, the antibodies of the invention comprise a heavychain comprising an amino acid sequence of one or more of SEQ ID NOs:188-190, 204, 205, 210, 1-3, 17-19, 33-35, 49-51, 113-115, 65-67, 81-83,97-99, 129-131, 145-147, 174-176, 161 or 162. For example, theantibodies of the invention comprise a heavy chain comprising SEQ ID NO:188 for CDRH1, SEQ ID NO: 189 for CDRH2, SEQ ID NO: 190 for CDRH3; SEQID NO: 188 for CDRH1, SEQ ID NO; 204 for CDRH2, SEQ ID NO: 205 forCDRH3; SEQ ID NO; 188 for CDRH1, SEQ ID NO: 189 for CDRH2, SEQ ID NO:210 for CDRH3; SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, SEQ IDNO: 3 for CDRH3; SEQ ID NO; 17 for CDRH1, SEQ ID NO; 18 for CDRH2, SEQID NO: 19 for CDRH3; SEQ ID NO: 33 for CDRH1, SEQ ID NO: 34 for CDRH2,SEQ ID NO: 35 for CDRH3; SEQ ID NO 49 for CHRH1, SEQ ID NO: 50 forCHRH2, SEQ ID NO: 51 for CDRH3; SEQ ID NO: 113 for CDRH1, SEQ ID NO: 114for CDRH2, SEQ ID NO: 115 for CDRH3; SEQ ID NO: 65 for CDRH1, SEQ ID NO:66 for CDRH2, SEQ ID NO: 67 for CDRH3; SEQ ID NO: 81 for CDRH1, SEQ IDNO 82 for CDRH2, SEQ ID NO: 83 for CDRH3; SEQ ID NO: 97 for CDRH1, SEQID NO: 98 for CDRH2, SEQ ID NO: 99 for CDRH3; SEQ ID NO: 129 for CDRH1,SEQ ID NO: 130 for CDRH2, SEQ ID NO: 131 for CDRH3; SEQ ID NO: 145 forCDRH1, SEQ ID NO: 146 for CDRH2, SEQ ID NO: 147 for CDRH3; SEQ ID NO:174 for CDRH1, SEQ ID NO: 175 for CDRH2, SEQ ID NO: 176 for CDRH3; andSEQ ID NO: 113 for CDRH1, SEQ ID NO: 161 for CDRH2, SEQ ID NO: 162 forCDRH3.

In yet another embodiment, the antibodies of the invention comprise alight chain comprising an amino acid sequence of one or more of SEQ IDNOs: 191-193, 4-6, 20-22, 36-38, 52-54, 116-118, 68-70, 84-86, 100-102,132-134, 148-150, 177, 178, 163, or 164. For example, the antibodies ofthe invention comprise a light chain comprising SEQ ID NO: 191 forCDRL1, SEQ ID NO: 192 for CDRL2; SEQ ID NO: 193 for CDRL3; SEQ ID NO: 4for CDRL1, SEQ ID NO: 5 for CDRL2 and SEQ ID NO: 6 for CDRL3; SEQ ID NO:20 for CDRL1, SEQ ID NO: 21 for CDRL2, SEQ ID NO: 22 for CDRL3; SEQ IDNO; 36 for CDRL1, SEQ ID NO: 37 for CDRL2, SEQ ID NO: 38 for CDRL3; SEQID NO: 52 for CDRL1, SEQ ID NO: 53 for CDRL2, SEQ ID NO: 54 for CDRL3;SEQ ID NO: 116 for CDRL1, SEQ ID NO: 117 for CDRL2, SEQ ID NO: 118 forCDRL3; SEQ ID NO: 68 for CDRL1, SEQ ID NO: 69 for CDRL2, SEQ ID NO: 70for CDRL3; SEQ ID NO 84 for CDRL1, SEQ ID NO: 85 for CDRL2, SEQ ID NO:86 for CDRL3; SEQ ID NO: 100 for CDRL1, SEQ ID NO: 101 for CDRL2, SEQ IDNO: 102 for CDRL3; SEQ ID NO: 132 for CDRL1, SEQ ID NO: 133 for CDRL2,SEQ ID NO: 134 for CDRL3; SEQ ID NO: 148 for CDRL1, SEQ ID NO: 149 forCDRL2, SEQ ID NO: 150 for CDRL3; SEQ ID NO: 177 for CDRL1, SEQ ID NO:149 for CDRL2, SEQ ID NO: 178 for CDRL3; SEQ ID NO: 163 for CDRL1, SEQID NO: 149 for CDRL2 and SEQ ID NO: 164 for CDRL3.

In still another embodiment, the antibodies of the invention comprise aheavy chain with an amino acid sequence that is at least 70% identicalto those of SEQ ID NOs: 200, 208, 212, 13, 29, 45, 61, 125, 77, 93, 109,141, 157, 184, or 170, and neutralize hCMV infection. In one embodiment,the antibody binds to an epitope in the hCMV UL128 protein and comprisesa heavy chain having an amino acid sequence that is at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% identical to the amino acid sequence of SEQID NO: 200, 208 or 212, and neutralizes hCMV infection. In oneembodiment, an antibody according to the invention comprises a heavychain having the sequence recited in SEQ ID NO: 200, 208 or 212, andneutralizes hCMV infection.

In another embodiment, the antibody binds to an epitope formed by thehCMV proteins UL130 and UL131A and comprises a heavy chain having anamino acid sequence that is at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the amino acid sequence of SEQ ID NO: 13, 29, 45, 61 or125, and neutralizes hCMV infection. In one embodiment, an antibodyaccording to the invention comprises a heavy chain having the sequencerecited in SEQ ID NO: 13, 29, 45, 61 or 125, and neutralizes hCMVinfection.

In yet another embodiment, the antibody binds to an epitope formed bythe hCMV proteins UL128, UL130 and UL131A and comprises a heavy chainhaving an amino acid sequence that is at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% identical to the amino acid sequence of SEQ ID NO: 77, 93,109, 141, 157, or 170, and neutralizes hCMV infection. In oneembodiment, an antibody according to the invention comprises a heavychain having the sequence recited in SEQ ID NO: 77, 93, 109, 141, 157,or 170, and neutralizes hCMV infection.

In a further embodiment, the antibody binds to an epitope formed by thehCMV proteins gH, gL, UL128 and UL130 and comprises a heavy chain havingan amino acid sequence that is at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the amino acid sequence of SEQ ID NO: 184, and neutralizeshCMV infection. In one embodiment, an antibody according to theinvention comprises a heavy chain having the sequence recited in SEQ IDNO: 184, and neutralizes hCMV infection.

In yet another embodiment, the antibodies of the invention comprise alight chain with an amino acid sequence that is at least 70% identicalto those of SEQ ID NOs: 201, 213, 14, 30, 46, 62, 126, 78, 94, 110, 142,158, 185, or 171, and neutralize hCMV infection.

In one embodiment, the antibody binds to an epitope in the hCMV UL128protein and comprises a light chain having an amino acid sequence thatis at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical to the amino acidsequence of SEQ ID NO: 201 or 213, and neutralizes hCMV infection. Inone embodiment, an antibody according to the invention comprises a lightchain having the sequence recited in SEQ ID NO: 201 or 213, andneutralizes hCMV infection.

In one embodiment, the antibody binds to an epitope formed by the hCMVproteins UL130 and UL131A and comprises a light chain having an aminoacid sequence that is at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% identicalto the amino acid sequence of SEQ ID NO: 14, 30, 46, 62 or 126, andneutralizes hCMV infection. In one embodiment, an antibody according tothe invention comprises a light chain having the sequence recited in SEQID NO: 14, 30, 46, 62 or 126, and neutralizes hCMV infection.

In another embodiment, the antibody binds to an epitope formed by thehCMV proteins UL128, UL130 and UL131A and comprises a light chain havingan amino acid sequence that is at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the amino acid sequence of SEQ ID NO: 78, 94, 110, 142,158, or 171, and neutralizes hCMV infection. In one embodiment, anantibody according to the invention comprises a light chain having thesequence recited in SEQ ID NO: 78, 94, 110, 142, 158, or 171, andneutralizes hCMV infection.

In a further embodiment, the antibody binds to an epitope formed by thehCMV proteins gH, gL, UL128 and UL130 and comprises a light chain havingan amino acid sequence that is at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the amino acid sequence of SEQ ID NO: 185, and neutralizeshCMV infection. In one embodiment, an antibody according to theinvention comprises a light chain having the sequence recited in SEQ IDNO: 185, and neutralizes hCMV infection.

In another embodiment, the antibodies or antibody fragments of theinvention comprise one or more heavy or light chain CDRs of theexemplary antibodies of the invention. In an exemplary embodiment, theantibodies or antibody fragments of the invention comprise an amino acidsequence selected from the group consisting of SEQ ID NOs: 316-321, 332,336-341, 278-283, 352, 296-301, 312, 232-236, 149, 216-221, 246-251,360, 361 and 262-267, and neutralize hCMV infection.

In yet another embodiment, the antibodies of the invention comprise aheavy chain comprising an amino acid sequence of one or more of SEQ IDNOs: 316-318, 332, 336-338, 278-280, 352, 296-298, 312, 232-234,216-218, 246-248, 360, 361 and 262-264. For example, the antibodies ofthe invention comprise a heavy chain comprising SEQ ID NO: 316 forCDRH1, SEQ ID NO: 317 for CDRH2, SEQ ID NO: 318 for CDRH3; SEQ ID NO:316 for CDRH1, SEQ ID NO: 317 for CDRH2, and SEQ ID NO: 332 for CDRH3;SEQ ID NO: 336 for CDRH1, SEQ ID NO: 337 for CDRH2, SEQ ID NO: 338 forCDRH3; SEQ ID NO: 278 for CDRH1, SEQ ID NO: 279 for CDRH2, SEQ ID NO:280 for CDRH3; SEQ ID NO: 352 for CDRH1, SEQ ID NO: 279 for CDRH2, SEQID NO: 280 for CDRH3; SEQ ID NO: 296 for CDRH1, SEQ ID NO: 297 forCDRH2, SEQ ID NO: 298 for CDRH3; SEQ ID NO: 296 for CDRH1, SEQ ID NO:312 for CDRH2, SEQ ID NO: 298 for CDRH3; SEQ ID NO: 232 for CDRH1, SEQID NO: 233 for CDRH2, SEQ ID NO: 234 for CDRH3; SEQ ID NO: 216 forCDRH1, SEQ ID NO: 217 for CDRH2, SEQ ID NO: 218 for CDRH3; SEQ ID NO:246 for CDRH1, SEQ ID NO: 247 for CDRH2, SEQ ID NO: 248 for CDRH3; andSEQ ID NO: 360 for CDRH1, SEQ ID NO: 279 for CDRH2, SEQ ID NO: 280 forCDRH3; and SEQ ID NO: 262 for CDRH1, SEQ ID NO: 263 for CDRH2, SEQ IDNO: 264 for CDRH3.

In still another embodiment, the antibodies of the invention comprise alight chain comprising an amino acid sequence of one or more of SEQ IDNOs: 319-321, 339-341, 281-283, 299-301, 149, 235, 236, 219-221,249-251, 265-267. For example, the antibodies of the invention comprisea light chain comprising SEQ ID NO: 319 for CDRL1, SEQ ID NO: 320 forCDRL2, SEQ ID NO: 321 for CDRL3; SEQ ID NO: 339 for CDRL1, SEQ ID NO:340 for CDRL2, SEQ ID NO: 341 for CDRL3; SEQ ID NO: 281 for CDRL1, SEQID NO: 282 for CDRL2, SEQ ID NO: 283 for CDRL3; SEQ ID NO: 299 forCDRL1, SEQ ID NO: 300 for CDRL2, SEQ ID NO: 301 for CDRL3; SEQ ID NO:235 for CDRL1, SEQ ID NO: 149 for CDRL2, SEQ ID NO: 236 for CDRL3; SEQID NO: 219 for CDRL1, SEQ ID NO: 220 for CDRL2, SEQ ID NO: 221 forCDRL3; SEQ ID NO: 249 for CDRL1, SEQ ID NO: 250 for CDRL2, SEQ ID NO:251 for CDRL3; and SEQ ID NO: 281 for CDRL1, SEQ ID NO: 282 for CDRL2,SEQ ID NO: 361 for CDRL3; and SEQ ID NO: 265 for CDRL1, SEQ ID NO: 266for CDRL2, SEQ ID NO: 267 for CDRL3.

In a further embodiment, the antibodies of the invention comprise aheavy chain with an amino acid sequence that is at least 70% identicalto those of SEQ ID NOs: 328, 334, 348, 290, 294, 357, 308, 314, 242,228, 258, 367 or 274, and neutralizes hCMV infection.

In one embodiment, the antibody binds to an epitope in the hCMV gBprotein and comprises a heavy chain having an amino acid sequence thatis at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical to the amino acidsequence of SEQ ID NO: 328, 334, 348, 290, 294, 308, 357, 314 or 367,and neutralizes hCMV infection. In one embodiment, an antibody accordingto the invention comprises a heavy chain having the sequence recited inSEQ ID NO: 328, 334, 348, 290, 294, 308, 357, 314 or 367 and neutralizeshCMV infection.

In another embodiment, the antibody binds to an epitope in the hCMV gHprotein and comprises a heavy chain having an amino acid sequence thatis at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical to the amino acidsequence of SEQ ID NO: 242, 228, or 258, and neutralizes hCMV infection.In one embodiment, an antibody according to the invention comprises aheavy chain having the sequence recited in SEQ ID NO: 242, 228, or 258,and neutralizes hCMV infection.

In another embodiment, the antibody binds to an epitope formed by thehCMV proteins gM and gN and comprises a heavy chain having an amino acidsequence that is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 98%, or at least 99% identical tothe amino acid sequence of SEQ ID NO: 274, and neutralizes hCMVinfection. In one embodiment, an antibody according to the inventioncomprises a heavy chain having the sequence recited in SEQ ID NO: 274,and neutralizes hCMV infection.

In yet another embodiment, the antibodies of the invention comprise alight chain with an amino acid sequence that is at least 70% identicalto those of SEQ ID NOs: 329, 349, 291, 309, 243, 229, 259, 368 or 275,and neutralize hCMV infection.

In one embodiment, the antibody binds to an epitope in the hCMV gBprotein and comprises a light chain having an amino acid sequence thatis at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical to the amino acidsequence of SEQ ID NO: 329, 349, 291, 309, or 368 and neutralizes hCMVinfection. In one embodiment, an antibody according to the inventioncomprises a light chain having the sequence recited in SEQ ID NO: 329,349, 291, 309 or 368, and neutralizes hCMV infection.

In another embodiment, the antibody binds to an epitope in the hCMV gHprotein and comprises a light chain having an amino acid sequence thatis at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical to the amino acidsequence of SEQ ID NO: 243, 229, or 259, and neutralizes hCMV infection.In one embodiment, an antibody according to the invention comprises alight chain having the sequence recited in SEQ ID NO: 243, 229, or 259,and neutralizes hCMV infection.

In another embodiment, the antibody binds to an epitope formed by thehCMV proteins gM and gN and comprises a light chain having an amino acidsequence that is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 98%, or at least 99% identical tothe amino acid sequence of SEQ ID NO: 275, and neutralizes hCMVinfection. In one embodiment, an antibody according to the inventioncomprises a light chain having the sequence recited in SEQ ID NO: 275,and neutralizes hCMV infection.

In one embodiment, the antibody of the invention is not MSL-109, 8F9,3E3 or R551A. In another embodiment, the antibody of the invention isnot 1F11, 2F4, 5A2 or 6G4, disclosed in U.S. application Ser. Nos.11/969,104 and 12/174,568.

Exemplary antibodies of the invention include, but are not limited to,15D8, 4N10, 10F7, 10P3, 4122, 8L13, 2C12, 8C15, 916, 7B13, 8J16, 8I21,7I13, 7H3, 6B4, 5F1, 10C6, 4H9, 2B11, 11B12, 13H11, 3G16 and 6L3.

Variants of 15D8 that neutralize hCMV infection consist of a heavy chainvariant having amino acid sequence recited in SEQ ID NO: 208 (“15D8variant 1”), and SEQ ID NO: 212 (“15D8 variant 2”), and a light chainhaving the amino acid sequence recited in SEQ ID NO: 213 (15D8 variant2). The nucleic acid sequences encoding the variant heavy chain variantsare recited in SEQ ID NO: 209 (15D8 variant 1) and SEQ ID NO: 214 (15D8variant 2). The nucleic acid encoding the variant light chain is recitedin SEQ ID NO: 215 (15D8 variant 2). Thus, antibodies comprising the 15D8variant heavy chains (SEQ ID NO: 208, 212) and variant light chain (SEQID NO: 213) that neutralize hCMV infection are included within the scopeof the invention.

As used herein, the term “15D8” is used to refer to any and/or allvariants of 15D8 that neutralize hCMV infection, for example, those withheavy chains corresponding to SEQ ID NO: 208 and 212 and light chainscorresponding to SEQ ID NO; 213.

A variant of 7H3 that neutralizes hCMV infection consists of a heavychain having the amino acid sequence recited in SEQ ID NO: 334 (“7H3variant 1”). The nucleic acid sequence encoding the variant heavy chainis recited in SEQ ID NO: 335. Thus, antibodies comprising the 7H3variant heavy chain (SEQ ID NO: 334) that neutralize hCMV infection areincluded within the scope of the invention.

As used herein, the term “7H3” is used to refer to any and/or allvariants of 7H3 that neutralize hCMV infection, for example, those withheavy chains corresponding to SEQ ID NO:334.

A variant of 5F1 that neutralizes hCMV infection consists of a heavychain having the amino acid sequence recited in SEQ ID NO: 294 (“5F1variant 1”). The nucleic acid sequence encoding the variant heavy chainis recited in SEQ ID NO: 295. Thus, antibodies comprising the 5F1variant heavy chain (SEQ ID NO: 294) that neutralize hCMV infection areincluded within the scope of the invention.

As used herein, the term “5F1” is used to refer to any and/or allvariants of 5F1 that neutralize hCMV infection, for example, those withheavy chains corresponding to SEQ ID NO:294.

A variant of 4H9 that neutralizes hCMV infection consists of a heavychain having the amino acid sequence recited in SEQ ID NO: 314 (“4H9variant 1”). The nucleic acid sequence encoding the variant heavy chainis recited in SEQ ID NO: 315. Thus, antibodies comprising the 4H9variant heavy chain (SEQ ID NO: 314), that neutralize hCMV infection areincluded within the scope of the invention.

As used herein, the term “4H9” is used to refer to any and/or allvariants of 4H9 that neutralize hCMV infection, for example, those withheavy chains corresponding to SEQ ID NO:314.

In one embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody 15D8 as listedin Table 1, and neutralizes hCMV infection in a human host. In anotherembodiment, an antibody of the invention, or antigen binding fragmentthereof, comprises all of the CDRs of antibody 15D8 variant 1 as listedin Table 1, and neutralizes hCMV infection in a human host. In anotherembodiment, an antibody of the invention, or antigen binding fragmentthereof, comprises all of the CDRs of antibody 15D8 variant 2 as listedin Table 1, and neutralizes hCMV infection in a human host. In yetanother embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody 8I21 as listedin Table 1, and neutralizes hCMV infection in a human host.

In yet another embodiment, an antibody of the invention, or antigenbinding fragment thereof, comprises all of the CDRs of antibody 4N10 aslisted in Table 1, and neutralizes hCMV infection in a human host. Inanother embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody 10F7 as listedin Table 1, and neutralizes hCMV infection in a human host. In anotherembodiment, an antibody of the invention, or antigen binding fragmentthereof, comprises all of the CDRs of antibody 10P3 as listed in Table1, and neutralizes hCMV infection in a human host. In anotherembodiment, an antibody of the invention, or antigen binding fragmentthereof, comprises all of the CDRs of antibody 4I22 as listed in Table1, and neutralizes hCMV infection in a human host. In anotherembodiment, an antibody of the invention, or antigen binding fragmentthereof, comprises all of the CDRs of antibody 8L13 as listed in Table1, and neutralizes hCMV infection in a human host.

In yet another embodiment, an antibody of the invention, or antigenbinding fragment thereof, comprises all of the CDRs of antibody 2C12 aslisted in Table 1, and neutralizes hCMV infection in a human host. Inanother embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody 8C15 as listedin Table 1, and neutralizes hCMV infection in a human host. In anotherembodiment, an antibody of the invention, or antigen binding fragmentthereof, comprises all of the CDRs of antibody 916 as listed in Table 1,and neutralizes hCMV infection in a human host. In another embodiment,an antibody of the invention, or antigen binding fragment thereof,comprises all of the CDRs of antibody 7B13 as listed in Table 1, andneutralizes hCMV infection in a human host. In another embodiment, anantibody of the invention, or antigen binding fragment thereof,comprises all of the CDRs of antibody 8J16 as listed in Table 1, andneutralizes hCMV infection in a human host. In another embodiment, anantibody of the invention, or antigen binding fragment thereof,comprises all of the CDRs of antibody 7I13 as listed in Table 1, andneutralizes hCMV infection in a human host.

In yet another embodiment, an antibody of the invention, or antigenbinding fragment thereof, comprises all of the CDRs of antibody 7H3 aslisted in Table 1, and neutralizes hCMV infection in a human host. Inanother embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody 7H3 variant 1 aslisted in Table 1, and neutralizes hCMV infection in a human host. Inanother embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody 6B4 as listed inTable 1, and neutralizes hCMV infection in a human host. In anotherembodiment, an antibody of the invention, or antigen binding fragmentthereof, comprises all of the CDRs of antibody 5F1 as listed in Table 1,and neutralizes hCMV infection in a human host. In another embodiment,an antibody of the invention, or antigen binding fragment thereof,comprises all of the CDRs of antibody 10C6 as listed in Table 1, andneutralizes hCMV infection in a human host. In another embodiment, anantibody of the invention, or antigen binding fragment thereof,comprises all of the CDRs of antibody 4H9 as listed in Table 1, andneutralizes hCMV infection in a human host. In another embodiment, anantibody of the invention, or antigen binding fragment thereof,comprises all of the CDRs of antibody 4H9 variant 1 as listed in Table1, and neutralizes hCMV infection in a human host. In anotherembodiment, an antibody of the invention, or antigen binding fragmentthereof, comprises all of the CDRs of antibody 2B11 as listed in Table1, and neutralizes hCMV infection in a human host.

In yet another embodiment, an antibody of the invention, or antigenbinding fragment thereof, comprises all of the CDRs of antibody 11B12 aslisted in Table 1, and neutralizes hCMV infection in a human host. Inanother embodiment, an antibody of the invention, or antigen bindingfragment thereof, comprises all of the CDRs of antibody 13H11 as listedin Table 1, and neutralizes hCMV infection in a human host. In anotherembodiment, an antibody of the invention, or antigen binding fragmentthereof, comprises all of the CDRs of antibody 3G16 as listed in Table1, and neutralizes hCMV infection in a human host. In yet anotherembodiment, an antibody of the invention, or antigen binding fragmentthereof, comprises all of the CDRs of antibody 6L3 as listed in Table 1,and neutralizes hCMV infection in a human host.

The invention further comprises an antibody, or fragment thereof, thatbinds to an epitope capable of binding to an antibody of the invention,or an antibody that competes with an antibody of the invention.

Antibodies of the invention also include hybrid antibody molecules thatcomprise one or more CDRs from an antibody of the invention and one ormore CDRs from another antibody to the same epitope. In one embodiment,such hybrid antibodies comprise three CDRs from an antibody of theinvention and three CDRs from another antibody to the same epitope.Exemplary hybrid antibodies comprise i) the three light chain CDRs froman antibody of the invention and the three heavy chain CDRs from anotherantibody to the same epitope, or ii) the three heavy chain CDRs from anantibody of the invention and the three light chain CDRs from anotherantibody to the same epitope.

In another aspect, the invention also includes nucleic acid sequencesencoding part or all of the light and heavy chains and CDRs of theantibodies of the present invention. In one embodiment, nucleic acidsequences according to the invention include nucleic acid sequenceshaving at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% identity to the nucleicacid encoding a heavy or light chain of an antibody of the invention. Inanother embodiment, a nucleic acid sequence of the invention has thesequence of a nucleic acid encoding a heavy or light chain CDR of anantibody of the invention. For example, a nucleic acid sequenceaccording to the invention comprises a sequence that is at least 75%identical to the nucleic acid sequences of SEQ ID NOs: 7-12, 15, 16,23-28, 31, 32, 39-44, 47, 48, 55-60, 63, 64, 71-76, 79, 80, 87-92, 95,96, 103-108, 111, 112, 119-124, 127, 128, 135-140, 143, 144, 151-156,159, 160, 165-169, 172, 173, 179-183, 186, 187, 194-199, 202, 203, 206,207, 209, 211, 214, 215, 222-227, 230, 231, 237-241, 244, 245, 252-257,260, 261, 268-273, 276, 277, 284-289, 292, 293, 295, 302-307, 310, 311,313, 315, 322-327, 330, 331, 333, 335, 342-347, 350, 351, 353-356, 358,359, 362-364, 365, 366, 369 and 370. In one embodiment, the nucleic acidsequence according to the invention comprises a sequence that is atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, or at least 99% identical to the nucleic acid sequences ofthe above listed SEQ ID NOs.

Due to the redundancy of the genetic code, variants of these sequenceswill exist that encode the same amino acid sequences. These variants areincluded within the scope of the invention.

Variant antibodies that neutralize hCMV infection are also includedwithin the scope of the invention. Thus, variants of the sequencesrecited in the application are also included within the scope of theinvention. Such variants include natural variants generated by somaticmutation in vivo during the immune response or in vitro upon culture ofimmortalized B cell clones. Alternatively, variants may arise due to thedegeneracy of the genetic code, as mentioned above or may be produceddue to errors in transcription or translation.

Further variants of the antibody sequences having improved affinityand/or potency may be obtained using methods known in the art and areincluded within the scope of the invention. For example, amino acidsubstitutions may be used to obtain antibodies with further improvedaffinity. Alternatively, codon optimisation of the nucleotide sequencemay be used to improve the efficiency of translation in expressionsystems for the production of the antibody. Further, polynucleotidescomprising a sequence optimized for antibody specificity or neutralizingactivity by the application of a directed evolution method to any of thenucleic acid sequences of the invention are also within the scope of theinvention.

In one embodiment variant antibody sequences that neutralize hCMVinfection may share 70% or more (i.e. 75%, 80%, 85%, 90%, 95%, 97%, 98%,99% or more) amino acid sequence identity with the sequences recited inthe application. In some embodiments such sequence identity iscalculated with regard to the full length of the reference sequence(i.e. the sequence recited in the application). In some furtherembodiments, percentage identity, as referred to herein, is asdetermined using BLAST version 2.1.3 using the default parametersspecified by the NCBI (the National Center for BiotechnologyInformation) [Blosum 62 matrix; gap open penalty=11 and gap extensionpenalty=1].

Further included within the scope of the invention are vectors, forexample expression vectors, comprising a nucleic acid sequence accordingto the invention. Cells transformed with such vectors are also includedwithin the scope of the invention. Examples of such cells include butare not limited to, eukaryotic cells, e.g. yeast cells, animal cells orplant cells. In one embodiment the cells are mammalian, e.g. human, CHO,HEK293T, PER.C6, NS0, myeloma or hybridoma cells.

The invention also relates to monoclonal antibodies that bind to anepitope capable of binding the antibodies of the invention, including,but not limited to, a monoclonal antibody selected from the groupconsisting of 15D8, 4N10, 10F7, 10P3, 4122, 8L13, 2C12, 8C15, 916, 7B13,8J16, 8I21, 7I13, 7H3, 6B4, 5F1, 10C6, 4H9, 11B12, 13H11, 3G16, 2B11 and6L3.

Monoclonal and recombinant antibodies are particularly useful inidentification and purification of the individual polypeptides or otherantigens against which they are directed. The antibodies of theinvention have additional utility in that they may be employed asreagents in immunoassays, radioimmunoassays (RIA) or enzyme-linkedimmunosorbent assays (ELISA). In these applications, the antibodies canbe labelled with an analytically-detectable reagent such as aradioisotope, a fluorescent molecule or an enzyme. The antibodies mayalso be used for the molecular identification and characterisation(epitope mapping) of antigens.

Antibodies of the invention can be coupled to a drug for delivery to atreatment site or coupled to a detectable label to facilitate imaging ofa site comprising cells of interest, such as cells infected with hCMV.Methods for coupling antibodies to drugs and detectable labels are wellknown in the art, as are methods for imaging using detectable labels.Labelled antibodies may be employed in a wide variety of assays,employing a wide variety of labels. Detection of the formation of anantibody-antigen complex between an antibody of the invention and anepitope of interest (an hCMV epitope) can be facilitated by attaching adetectable substance to the antibody. Suitable detection means includethe use of labels such as radionuclides, enzymes, coenzymes,fluorescers, chemiluminescers, chromogens, enzyme substrates orco-factors, enzyme inhibitors, prosthetic group complexes, freeradicals, particles, dyes, and the like. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material isluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S, or ³H. Such labeled reagents may be used in avariety of well-known assays, such as radioimmunoassays, enzymeimmunoassays, e.g., ELISA, fluorescent immunoassays, and the like. Seefor example, references 15-18.

An antibody according to the invention may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent, or aradioactive metal ion or radioisotope. Examples of radioisotopesinclude, but are not limited to, I-131, I-123, I-125, Y-90, Re-188,Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and thelike. Such antibody conjugates can be used for modifying a givenbiological response; the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin.

Techniques for conjugating such therapeutic moiety to antibodies arewell known. See, for example, Amon et al. (1985) “Monoclonal Antibodiesfor Immunotargeting of Drugs in Cancer Therapy,” in MonoclonalAntibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.),pp. 243-256; ed. Hellstrom et al. (1987) “Antibodies for Drug Delivery,”in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker,Inc.), pp. 623-653; Thorpe (1985) “Antibody Carriers of Cytotoxic Agentsin Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biologicaland Clinical Applications, ed. Pinchera et al. pp. 475-506 (EditriceKurtis, Milano, Italy, 1985); “Analysis, Results, and Future Prospectiveof the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy,” inMonoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin etal. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al.(1982) Immunol. Rev. 62:119-158.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described in reference 19. Inaddition, linkers may be used between the labels and the antibodies ofthe invention [20]. Antibodies or, antigen-binding fragments thereof maybe directly labelled with radioactive iodine, indium, yttrium, or otherradioactive particle known in the art [21]. Treatment may consist of acombination of treatment with conjugated and non-conjugated antibodiesadministered simultaneously or subsequently [22, 23].

Antibodies of the invention may also be attached to a solid support.

Additionally, antibodies of the invention, or functional antibodyfragments thereof, can be chemically modified by covalent conjugation toa polymer to, for example, increase their circulating half-life, forexample. Examples of polymers, and methods to attach them to peptides,are shown in references 24-27. In some embodiments the polymers may beselected from polyoxyethylated polyols and polyethylene glycol (PEG).PEG is soluble in water at room temperature and has the general formula:R(O—CH₂—CH₂)_(n)O—R where R can be hydrogen, or a protective group suchas an alkyl or alkanol group. In one embodiment the protective group mayhave between 1 and 8 carbons. In a further embodiment the protectivegroup is methyl. The symbol n is a positive integer. In one embodiment nis between 1 and 1,000. In another embodiment n is between 2 and 500. Inone embodiment the PEG has an average molecular weight between 1,000 and40,000. In a further embodiment the PEG has a molecular weight between2,000 and 20,000. In yet a further embodiment the PEG has a molecularweight of between 3,000 and 12,000. In one embodiment PEG has at leastone hydroxy group. In another embodiment the PEG has a terminal hydroxygroup. In yet another embodiment it is the terminal hydroxy group whichis activated to react with a free amino group on the inhibitor. However,it will be understood that the type and amount of the reactive groupsmay be varied to achieve a covalently conjugated PEG/antibody of thepresent invention.

Water-soluble polyoxyethylated polyols are also useful in the presentinvention. They include polyoxyethylated sorbitol, polyoxyethylatedglucose, polyoxyethylated glycerol (POG), and the like. In oneembodiment. POG is used. Without being bound by any theory, because theglycerol backbone of polyoxyethylated glycerol is the same backboneoccurring naturally in, for example, animals and humans in mono-, di-,triglycerides, this branching would not necessarily be seen as a foreignagent in the body. In some embodiments POG has a molecular weight in thesame range as PEG. The structure for POG is shown in reference 28, and adiscussion of POG/IL-2 conjugates is found in reference 24.

Another drug delivery system that can be used for increasing circulatoryhalf-life is the liposome. Methods of preparing liposome deliverysystems are discussed in references 29, 30 and 31. Other drug deliverysystems are known in the art and are described in, for example,references 32 and 33.

Antibodies of the invention may be provided in purified form. Typically,the antibody will be present in a composition that is substantially freeof other polypeptides e.g. where less than 90% (by weight), usually lessthan 60% and more usually less than 50% of the composition is made up ofother polypeptides.

Antibodies of the invention may be immunogenic in non-human (orheterologous) hosts e.g. in mice. In particular, the antibodies may havean idiotope that is immunogenic in non-human hosts, but not in a humanhost. Antibodies of the invention for human use include those thatcannot be easily isolated from hosts such as mice, goats, rabbits, rats,non-primate mammals, etc. and cannot generally be obtained byhumanisation or from xeno-mice.

Antibodies of the invention can be of any isotype (e.g. IgA, IgG, IgMi.e. an α, γ or μ heavy chain), but will generally be IgG. Within theIgG isotype, antibodies may be IgG1, IgG2, IgG3 or IgG4 subclass.Antibodies of the invention may have a κ or a λ light chain.

Production of Antibodies

Monoclonal antibodies according to the invention can be made by anymethod known in the art. The general methodology for making monoclonalantibodies using hybridoma technology is well known [34, 35].Preferably, the alternative EBV immortalisation method described inreference 36 is used.

Using the method described in reference 36, B cells producing theantibody of the invention can be transformed with EBV in the presence ofa polyclonal B cell activator. Transformation with EBV is a standardtechnique and can easily be adapted to include polyclonal B cellactivators.

Additional stimulants of cellular growth and differentiation mayoptionally be added during the transformation step to further enhancethe efficiency. These stimulants may be cytokines such as IL-2 andIL-15. In one aspect, IL-2 is added during the immortalisation step tofurther improve the efficiency of immortalisation, but its use is notessential.

The immortalised B cells produced using these methods can then becultured using methods known in the art and antibodies isolatedtherefrom.

The antibodies of the invention can also be made by culturing singleplasma cells in microwell culture plates using the method described inUK Patent Application 0819376.5. Further, from single plasma cellcultures, RNA can be extracted and single cell PCR can be performedusing methods known in the art. The VH and VL regions of the antibodiescan be amplified by RT-PCR, sequenced and cloned into an expressionvector that is then transfected into HEK293T cells or other host cells.The cloning of nucleic acid in expression vectors, the transfection ofhost cells, the culture of the transfected host cells and the isolationof the produced antibody can be done using any methods known to one ofskill in the art.

Monoclonal antibodies may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Techniques for purification ofmonoclonal antibodies, including techniques for producingpharmaceutical-grade antibodies, are well known in the art.

Fragments of the monoclonal antibodies of the invention can be obtainedfrom the monoclonal antibodies by methods that include digestion withenzymes, such as pepsin or papain, and/or by cleavage of disulfide bondsby chemical reduction. Alternatively, fragments of the monoclonalantibodies can be obtained by cloning and expression of part of thesequences of the heavy or light chains. Antibody “fragments” may includeFab, Fab′, F(ab′)₂ and Fv fragments. The invention also encompassessingle-chain Fv fragments (scFv) derived from the heavy and light chainsof a monoclonal antibody of the invention e.g. the invention includes ascFv comprising the CDRs from an antibody of the invention. Alsoincluded are heavy or light chain monomers and dimers as well as singlechain antibodies, e.g. single chain Fv in which the heavy and lightchain variable domains are joined by a peptide linker.

Standard techniques of molecular biology may be used to prepare DNAsequences coding for the antibodies or fragments of the antibodies ofthe present invention. Desired DNA sequences may be synthesisedcompletely or in part using oligonucleotide synthesis techniques.Site-directed mutagenesis and polymerase chain reaction (PCR) techniquesmay be used as appropriate.

Any suitable host cell/vector system may be used for expression of theDNA sequences encoding the antibody molecules of the present inventionor fragments thereof. Bacterial, for example E. coli, and othermicrobial systems may be used, in part, for expression of antibodyfragments such as Fab and F(ab′)₂ fragments, and especially Fv fragmentsand single chain antibody fragments, for example, single chain Fvs.Eukaryotic, e.g. mammalian, host cell expression systems may be used forproduction of larger antibody molecules, including complete antibodymolecules. Suitable mammalian host cells include CHO, HEK293T, PER.C6,NS0, myeloma or hybridoma cells.

The present invention also provides a process for the production of anantibody molecule according to the present invention comprisingculturing a host cell comprising a vector of the present invention underconditions suitable for leading to expression of protein from DNAencoding the antibody molecule of the present invention, and isolatingthe antibody molecule.

The antibody molecule may comprise only a heavy or light chainpolypeptide, in which case only a heavy chain or light chain polypeptidecoding sequence needs to be used to transfect the host cells. Forproduction of products comprising both heavy and light chains, the cellline may be transfected with two vectors, a first vector encoding alight chain polypeptide and a second vector encoding a heavy chainpolypeptide. Alternatively, a single vector may be used, the vectorincluding sequences encoding light chain and heavy chain polypeptides.

Alternatively, antibodies according to the invention may be produced byi) expressing a nucleic acid sequence according to the invention in acell, and ii) isolating the expressed antibody product. Additionally,the method may include iii) purifying the antibody.

Screening and Isolation of B Cells

Transformed B cells may be screened for those producing antibodies ofthe desired antigen specificity, and individual B cell clones may thenbe produced from the positive cells.

The screening step may be carried out by ELISA, by staining of tissuesor cells (including transfected cells), a neutralisation assay or one ofa number of other methods known in the art for identifying desiredantigen specificity. The assay may select on the basis of simple antigenrecognition, or may select on the additional basis of a desired functione.g. to select neutralizing antibodies rather than just antigen-bindingantibodies, to select antibodies that can change characteristics oftargeted cells, such as their signalling cascades, their shape, theirgrowth rate, their capability of influencing other cells, their responseto the influence by other cells or by other reagents or by a change inconditions, their differentiation status, etc.

The cloning step for separating individual clones from the mixture ofpositive cells may be carried out using limiting dilution,micromanipulation, single cell deposition by cell sorting or anothermethod known in the art.

The immortalised B cell clones of the invention can be used in variousways e.g. as a source of monoclonal antibodies, as a source of nucleicacid (DNA or mRNA) encoding a monoclonal antibody of interest, forresearch, etc.

The invention provides a composition comprising immortalised B memorycells, wherein the cells produce antibodies with high neutralizingpotency specific for hCMV, and wherein the antibodies are produced at ≧5pg per cell per day. The invention also provides a compositioncomprising clones of an immortalised B memory cell, wherein the clonesproduce a monoclonal antibody with a high affinity specific for hCMV,and wherein the antibody is produced at ≧5 pg per cell per day.Preferably said clones produce a monoclonal antibody with a high potencyin neutralizing hCMV infection.

Exemplary immortalised B cell clone according to the invention include,but are not limited to, 15D8, 4N10, 10F7, 10P3, 4122, 8L13, 2C12, 8C15,916, 7B13, 8J16, 8I21, 7I13, 7H3, 6B4, 5F1, 10C6, 4H9, 11B12, 13H11,3G16, 2B11 and 6L3.

Epitopes

As mentioned above, the antibodies of the invention can be used to mapthe epitopes to which they bind. The inventors have discovered that theseveral antibodies neutralizing hCMV infection of endothelial cells,epithelial cells, retinal cells and dendritic cells, are directedtowards epitopes in the hCMV UL128 protein, epitopes formed by the hCMVproteins UL130 and UL131A, epitopes formed by the hCMV proteins UL128,UL130 and UL131A, epitopes formed by the hCMV proteins gH, gL, UL128 andUL130, gB, gH, or epitopes formed by the hCMV proteins gM and gN. Theepitopes to which the antibodies of the invention bind may be linear(continuous) or conformational (discontinuous) and formed by a singlehCMV protein or by the combination of 2, 3 or more hCMV proteins.

The epitopes recognised by the antibodies of the present invention mayhave a number of uses. The epitope and mimotopes thereof in purified orsynthetic form can be used to raise immune responses (i.e. as a vaccine,or for the production of antibodies for other uses) or for screeningpatient serum for antibodies that immunoreact with the epitope ormimotopes thereof. In one embodiment such an epitope or mimotope, orantigen comprising such an epitope or mimotope may be used as a vaccinefor raising an immune response. The antibodies and antibody fragments ofthe invention can also be used in a method of monitoring the quality ofvaccines. In particular the antibodies can be used to check that theantigen in a vaccine contains the specific immunogenic epitope in thecorrect conformation.

The epitope may also be useful in screening for ligands that bind tosaid epitope. Such ligands, include but are not limited to antibodies;including those from camels, sharks and other species, fragments ofantibodies, peptides, phage display technology products, aptamers,adnectins or fragments of other viral or cellular proteins, may blockthe epitope and so prevent infection. Such ligands are encompassedwithin the scope of the invention.

Recombinant Expression

The immortalised B memory cells of the invention may also be used as asource of nucleic acid for the cloning of antibody genes for subsequentrecombinant expression. Expression from recombinant sources is morecommon for pharmaceutical purposes than expression from B cells orhybridomas e.g. for reasons of stability, reproducibility, culture ease,etc.

Thus the invention provides a method for preparing a recombinant cell,comprising the steps of: (i) obtaining one or more nucleic acids (e.g.heavy and/or light chain genes) from the B cell clone that encodes theantibody of interest; and (ii) inserting the nucleic acid into anexpression host in order to permit expression of the antibody ofinterest in that host.

Similarly, the invention provides a method for preparing a recombinantcell, comprising the steps of: (i) sequencing nucleic acid(s) from the Bcell clone that encodes the antibody of interest; and (ii) using thesequence information from step (i) to prepare nucleic acid(s) forinsertion into an expression host in order to permit expression of theantibody of interest in that host. The nucleic acid may, but need not,be manipulated between steps (i) and (ii) to introduce restrictionsites, to change codon usage, and/or to optimise transcription and/ortranslation regulatory sequences.

The invention also provides a method of preparing a recombinant cell,comprising the step of transforming a host cell with one or more nucleicacids that encode a monoclonal antibody of interest, wherein the nucleicacids are nucleic acids that were derived from an immortalised B cellclone of the invention. Thus the procedures for first preparing thenucleic acid(s) and then using it to transform a host cell can beperformed at different times by different people in different places(e.g. in different countries).

These recombinant cells of the invention can then be used for expressionand culture purposes. They are particularly useful for expression ofantibodies for large-scale pharmaceutical production. They can also beused as the active ingredient of a pharmaceutical composition. Anysuitable culture techniques can be used, including but not limited tostatic culture, roller bottle culture, ascites fluid, hollow-fiber typebioreactor cartridge, modular minifermenter, stirred tank, microcarrierculture, ceramic core perfusion, etc.

Methods for obtaining and sequencing immunoglobulin genes from B cellsare well known in the art (e.g. see reference 37).

The expression host is preferably a eukaryotic cell, including yeast andanimal cells, particularly mammalian cells (e.g. CHO cells, NS0 cells,human cells such as PER.C6 [Crucell; reference 38] or HKB-11 [Bayer;references 39 & 40] cells, myeloma cells [41 & 42], etc.), as well asplant cells. Preferred expression hosts can glycosylate the antibody ofthe invention, particularly with carbohydrate structures that are notthemselves immunogenic in humans. In one embodiment the expression hostmay be able to grow in serum-free media. In a further embodiment theexpression host may be able to grow in culture without the presence ofanimal-derived products.

The expression host may be cultured to give a cell line.

The invention provides a method for preparing one or more nucleic acidmolecules (e.g. heavy and light chain genes) that encode an antibody ofinterest, comprising the steps of: (i) preparing an immortalised B cellclone according to the invention; (ii) obtaining from the B cell clonenucleic acid that encodes the antibody of interest. The invention alsoprovides a method for obtaining a nucleic acid sequence that encodes anantibody of interest, comprising the steps of: (i) preparing animmortalised B cell clone according to the invention; (ii) sequencingnucleic acid from the B cell clone that encodes the antibody ofinterest.

The invention also provides a method of preparing nucleic acidmolecule(s) that encodes an antibody of interest, comprising the step ofobtaining the nucleic acid from a B cell clone that was obtained from atransformed B cell of the invention. Thus the procedures for firstobtaining the B cell clone and then preparing nucleic acid(s) from itcan be performed at very different times by different people indifferent places (e.g. in different countries).

The invention provides a method for preparing an antibody (e.g. forpharmaceutical use), comprising the steps of: (i) obtaining and/orsequencing one or more nucleic acids (e.g. heavy and light chain genes)from the selected B cell clone expressing the antibody of interest; (ii)inserting the nucleic acid(s) into or using the nucleic acid(s) toprepare an expression host that can express the antibody of interest;(iii) culturing or sub-culturing the expression host under conditionswhere the antibody of interest is expressed; and, optionally, (iv)purifying the antibody of the interest.

The invention also provides a method of preparing an antibody comprisingthe steps of: culturing or sub-culturing an expression host cellpopulation under conditions where the antibody of interest is expressedand, optionally, purifying the antibody of the interest, wherein saidexpression host cell population has been prepared by (i) providingnucleic acid(s) encoding a selected B cell the antibody of interest thatis produced by a population of B memory lymphocytes prepared asdescribed above, (ii) inserting the nucleic acid(s) into an expressionhost that can express the antibody of interest, and (iii) culturing orsub-culturing expression hosts comprising said inserted nucleic acids toproduce said expression host cell population. Thus the procedures forfirst preparing the recombinant expression host and then culturing it toexpress antibody can be performed at very different times by differentpeople in different places (e.g. in different countries).

Further, cell lines expressing exemplary antibodies of the invention,4N10, 2C12, 8C15, 8I21, 6B4, 10C6, 4H9, 11B12, 3G16, and 6L3 weredeposited with the Advanced Biotechnology Center (ABC), Largo RossanaBenzi 10, 16132 Genoa (Italy), under the terms of the Budapest Treaty,on Jul. 9, 2008, (under Accession Numbers PD 08009, PD 08007, PD 08006,PD 08005, PD 08004, PD 08014, PD 08013, PD 08011, PD 08012, and PD08010, respectively) and an immortalized B cell line expressing 7H3 wasdeposited on Jul. 16, 2008 under Accession Number PD 08017. An antibody,or an antigen binding fragment thereof, expressed from the above celllines as well as antibodies, and antigen binding fragments thereof, withthe same amino acid sequence as those expressed from the above celllines are also considered to be within the scope of the invention.

These deposits are provided for the convenience of those skilled in theart and are neither an admission that such deposits are required topractice the invention nor that equivalent embodiments are not withinthe skill of the art in view of the present disclosure. The publicavailability of these deposits is not a grant of a license to make, useor sell the deposited materials under this or any other patents. Thenucleic acid sequences of the deposited materials are incorporated inthe present disclosure by reference and are controlling if in conflictwith any sequence described herein.

Pharmaceutical Compositions

The invention provides a pharmaceutical composition containing theantibodies and/or antibody fragments of the invention and/or nucleicacid encoding such antibodies and/or immortalised B cells that expresssuch antibodies and/or the epitopes recognised by the antibodies of theinvention. A pharmaceutical composition may also contain apharmaceutically acceptable carrier to allow administration. The carriershould not itself induce the production of antibodies harmful to theindividual receiving the composition and should not be toxic. Suitablecarriers may be large, slowly metabolised macromolecules such asproteins, polypeptides, liposomes, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers andinactive virus particles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the patient.

Within the scope of the invention, forms of administration may includethose forms suitable for parenteral administration, e.g. by injection orinfusion, for example by bolus injection or continuous infusion. Wherethe product is for injection or infusion, it may take the form of asuspension, solution or emulsion in an oily or aqueous vehicle and itmay contain formulatory agents, such as suspending, preservative,stabilising and/or dispersing agents. Alternatively, the antibodymolecule may be in dry form, for reconstitution before use with anappropriate sterile liquid.

Once formulated, the compositions of the invention can be administereddirectly to the subject. In one embodiment the compositions are adaptedfor administration to human subjects.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intraperitoneal,intrathecal, intraventricular, transdermal, transcutaneous, topical,subcutaneous, intranasal, enteral, sublingual, intravaginal or rectalroutes. Hyposprays may also be used to administer the pharmaceuticalcompositions of the invention. Typically, the therapeutic compositionsmay be prepared as injectables, either as liquid solutions orsuspensions. Solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection may also be prepared.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Dosage treatmentmay be a single dose schedule or a multiple dose schedule. Knownantibody-based pharmaceuticals provide guidance relating to frequency ofadministration e.g. whether a pharmaceutical should be delivered daily,weekly, monthly, etc. Frequency and dosage may also depend on theseverity of symptoms.

Compositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. Solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared (e.g. a lyophilised composition, like Synagis™ and Herceptin™,for reconstitution with sterile water containing a preservative). Thecomposition may be prepared for topical administration e.g. as anointment, cream or powder. The composition may be prepared for oraladministration e.g. as a tablet or capsule, as a spray, or as a syrup(optionally flavoured). The composition may be prepared for pulmonaryadministration e.g. as an inhaler, using a fine powder or a spray. Thecomposition may be prepared as a suppository or pessary. The compositionmay be prepared for nasal, aural or ocular administration e.g. as drops.The composition may be in kit form, designed such that a combinedcomposition is reconstituted just prior to administration to a patient.For example, a lyophilised antibody can be provided in kit form withsterile water or a sterile buffer.

It will be appreciated that the active ingredient in the compositionwill be an antibody molecule, an antibody fragment or variants andderivatives thereof. As such, it will be susceptible to degradation inthe gastrointestinal tract. Thus, if the composition is to beadministered by a route using the gastrointestinal tract, thecomposition will need to contain agents which protect the antibody fromdegradation but which release the antibody once it has been absorbedfrom the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Gennaro (2000) Remington: The Science and Practice ofPharmacy, 20th edition, ISBN: 0683306472.

Pharmaceutical compositions of the invention generally have a pH between5.5 and 8.5, in some embodiments this may be between 6 and 8, and infurther embodiments about 7. The pH may be maintained by the use of abuffer. The composition may be sterile and/or pyrogen free. Thecomposition may be isotonic with respect to humans. In one embodimentpharmaceutical compositions of the invention are supplied inhermetically-sealed containers.

Pharmaceutical compositions will include an effective amount of one ormore antibodies of the invention and/or one or more immortalised B cellsof the invention and/or a polypeptide comprising an epitope that bindsan antibody of the invention i.e. an amount that is sufficient to treat,ameliorate, or prevent a desired disease or condition, or to exhibit adetectable therapeutic effect. Therapeutic effects also includereduction in physical symptoms. The precise effective amount for anyparticular subject will depend upon their size and health, the natureand extent of the condition, and the therapeutics or combination oftherapeutics selected for administration. The effective amount for agiven situation is determined by routine experimentation and is withinthe judgment of a clinician. For purposes of the present invention, aneffective dose will generally be from about 0.01 mg/kg to about 50mg/kg, or about 0.05 mg/kg to about 10 mg/kg of the compositions of thepresent invention in the individual to which it is administered. Knownantibody-based pharmaceuticals provide guidance in this respect e.g.Herceptin™ is administered by intravenous infusion of a 21 mg/mlsolution, with an initial loading dose of 4 mg/kg body weight and aweekly maintenance dose of 2 mg/kg body weight; Rituxan™ is administeredweekly at 375 mg/m²; etc.

In one embodiment compositions can include more than one (e.g. 2, 3, 4,5, etc.) antibody of the invention to provide an additive or synergistictherapeutic effect. In a further embodiment the composition may compriseone or more (e.g. 2, 3, 4, 5, etc.) antibody of the invention and one ormore (e.g. 2, 3, 4, 5, etc.) additional antibodies that neutralize hCMVinfection.

For example, one antibody may bind to an epitope in the hCMV UL128protein, an epitope formed by the hCMV proteins UL130 and UL131A, anepitope formed by the hCMV proteins UL128, UL130 and UL131A, an epitopeformed by the hCMV proteins gH, gL, UL128 and UL130, an epitope in thehCMV gB protein, an epitope in the hCMV gH protein, or an epitope formedby the hCMV proteins gM and gN, while another may bind to a differentepitope in the hCMV UL128 protein, an epitope formed by UL130 andUL131A, an epitope formed by UL128, UL130 and UL131A, an epitope formedby gH, gL, UL128 and UL130, gB, gH, gL, gM, gN, gO, or an epitope formedby gM and gN. Without being bound to any theory, one antibody may betargeted to the mechanism that mediates infection of fibroblasts, whilethe other antibody may be targeted to the mechanism that mediatesinfection of endothelial cells. For optimal clinical effect it may wellbe advantageous to address both mechanisms of hCMV infection andmaintenance.

In one embodiment, the invention provides a pharmaceutical compositioncomprising two or more antibodies, wherein the first antibody isspecific for a first UL128 epitope, and the second antibody is specificfor a second UL128 epitope, a combination of UL130 and UL131A, acombination of UL128, UL130 and UL131A, a combination of gH, gL, UL128and UL130, gB, gH, gL, gM, gN, gO, or a combination of gM and gN.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising two or more antibodies, wherein the firstantibody is specific for a first epitope on a combination of UL130 and131A, and the second antibody is specific for UL128, a second epitope ona combination of UL130 and 131A, a combination of UL128, UL130 andUL131A, a combination of gH, gL, UL128 and UL130, gB, gH, gL, gM, gN,gO, or a combination of gM and gN.

In yet another embodiment, the invention provides a pharmaceuticalcomposition comprising two or more antibodies, wherein the firstantibody is specific for a first epitope on a combination of UL128,UL130 and 131A, and the second antibody is specific for UL128, acombination of UL130 and UL131A, a second epitope on a combination ofUL128, UL130 and 131A, a combination of gH, gL, UL128 and UL130, gB, gH,gL, gM, gN, gO, or a combination of gM and gN.

In still another embodiment, the invention provides a pharmaceuticalcomposition comprising two or more antibodies, wherein the firstantibody is specific for a first epitope on a combination of gH, gL,UL128, UL130 and UL131A, and the second antibody is specific for UL128,a combination of UL130 and UL131A, a combination of UL128, UL130 and131A, a second epitope on a combination of gH, gL, UL128 and UL130, gB,gH, gL, gM, gN, gO, or a combination of gM and gN.

In a further embodiment, the invention provides a pharmaceuticalcomposition comprising two or more antibodies, wherein the firstantibody is specific for a first gB epitope, and the second antibody isspecific for UL128, a combination of UL130 and UL131A, a combination ofUL128, UL130 and UL131A, a combination of gH, gL, UL128 and UL130, asecond gB epitope, gH, gL, gM, gN, gO, or a combination of gM and gN.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising two or more antibodies, wherein the firstantibody is specific for a first gH epitope, and the second antibody isspecific for UL128, a combination of UL130 and UL131A, a combination ofUL128, UL130 and UL131A, a combination of gH, gL, UL128 and UL130, gB, asecond gH epitope, gL, gM, gN, gO, or a combination of gM and gN.

In yet another embodiment, the invention provides a pharmaceuticalcomposition comprising two or more antibodies, wherein the firstantibody is specific for a first epitope on a combination of gM and gN,and the second antibody is specific for UL128, a combination of UL130and UL131A, a combination of UL128, UL130 and UL131A, a combination ofgH, gL, UL128 and UL130, gB, gH, gL, gM, gN, gO, or a second epitope ona combination of gM and gN.

Exemplary antibodies of the invention for use in a pharmaceuticalcomposition that bind to an epitope in the hCMV UL128 protein include,but are not limited to, 15D8. Exemplary antibodies of the invention foruse in a pharmaceutical composition that bind an epitope formed by thehCMV proteins UL130 and UL131A include, but are not limited to, 4N10,10F7, 10P3, 4122, 8L13, 1F11, 2F4 and 5A2 (see U.S. application Ser. No.11/969,104, filed Jan. 3, 2008). Exemplary antibodies of the inventionfor use in a pharmaceutical composition that bind an epitope formed bythe hCMV proteins UL128, UL130 and UL131A include, but are not limitedto, 2C12, 7B13, 7I13, 8C15, 8J16, 916, and 6G4 (see U.S. applicationSer. No. 12/174,568, filed Jul. 16, 2008). Exemplary antibodies of theinvention for use in a pharmaceutical composition that bind an epitopeformed by the hCMV proteins gH, gL, UL128 and UL130 include, but are notlimited to, 8I21. Exemplary antibodies of the invention for use in apharmaceutical composition that bind to an epitope in the hCMV gBprotein include, but are not limited to, 7H3, 10C6, 5F1, 6B4, 4H9 and2B11. Exemplary antibodies of the invention for use in a pharmaceuticalcomposition that bind to an epitope in the hCMV gH protein include, butare not limited to, 11B12, 13H11, and 3G16. Exemplary antibodies of theinvention for use in a pharmaceutical composition that bind an epitopeformed by the hCMV proteins gM and gN include, but are not limited to,6L3. The invention further provides a pharmaceutical compositioncomprising two or more antibodies, wherein the first antibody is anantibody or antibody fragment of the invention and the second antibodyis an antibody now known in the art, or later discovered, thatneutralises hCMV infection. Examples of such antibodies include, but arenot limited to MSL-109, 8F9 or 3E3.

In one embodiment, the invention provides a pharmaceutical compositioncomprising the antibody 15D8 or an antigen binding fragment thereof, anda pharmaceutically acceptable carrier. In another embodiment, theinvention provides a pharmaceutical composition comprising the antibody15D8 variant for an antigen binding fragment thereof, and apharmaceutically acceptable carrier. In another embodiment, theinvention provides a pharmaceutical composition comprising the antibody15D8 variant 2 or an antigen binding fragment thereof, and apharmaceutically acceptable carrier. In another embodiment, theinvention provides a pharmaceutical composition comprising the antibody8I21 or an antigen binding fragment thereof, and a pharmaceuticallyacceptable carrier.

In yet another embodiment, the invention provides a pharmaceuticalcomposition comprising the antibody 2C12 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody 8C15 or an antigen binding fragment thereof, anda pharmaceutically acceptable carrier. In another embodiment, theinvention provides a pharmaceutical composition comprising the antibody916 or an antigen binding fragment thereof, and a pharmaceuticallyacceptable carrier. In another embodiment, the invention provides apharmaceutical composition comprising the antibody 7B13 or an antigenbinding fragment thereof, and a pharmaceutically acceptable carrier. Inanother embodiment, the invention provides a pharmaceutical compositioncomprising the antibody 8J16 or an antigen binding fragment thereof, anda pharmaceutically acceptable carrier. In another embodiment, theinvention provides a pharmaceutical composition comprising the antibody7I13 or an antigen binding fragment thereof, and a pharmaceuticallyacceptable carrier.

In yet another embodiment, the invention provides a pharmaceuticalcomposition comprising the antibody 4N10 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody 10F7 or an antigen binding fragment thereof, anda pharmaceutically acceptable carrier. In another embodiment, theinvention provides a pharmaceutical composition comprising the antibody10P3 or an antigen binding fragment thereof, and a pharmaceuticallyacceptable carrier. In another embodiment, the invention provides apharmaceutical composition comprising the antibody 4I22 or an antigenbinding fragment thereof, and a pharmaceutically acceptable carrier. Inanother embodiment, the invention provides a pharmaceutical compositioncomprising the antibody 8L13 or an antigen binding fragment thereof, anda pharmaceutically acceptable carrier.

In yet another embodiment, the invention provides a pharmaceuticalcomposition comprising the antibody 7H3 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody 7H3 variant 1 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody 10C6 or an antigen binding fragment thereof, anda pharmaceutically acceptable carrier. In another embodiment, theinvention provides a pharmaceutical composition comprising the antibody5F1 or an antigen binding fragment thereof, and a pharmaceuticallyacceptable carrier. In another embodiment, the invention provides apharmaceutical composition comprising the antibody 6B4 or an antigenbinding fragment thereof, and a pharmaceutically acceptable carrier. Inanother embodiment, the invention provides a pharmaceutical compositioncomprising the antibody 4H9 or an antigen binding fragment thereof, anda pharmaceutically acceptable carrier. In another embodiment, theinvention provides a pharmaceutical composition comprising the antibody4H9 variant 1 or an antigen binding fragment thereof, and apharmaceutically acceptable carrier. In another embodiment, theinvention provides a pharmaceutical composition comprising the antibody2B11 or an antigen binding fragment thereof, and a pharmaceuticallyacceptable carrier.

In yet another embodiment, the invention provides a pharmaceuticalcomposition comprising the antibody 13H11 or an antigen binding fragmentthereof, and a pharmaceutically acceptable carrier. In anotherembodiment, the invention provides a pharmaceutical compositioncomprising the antibody 11B12 or an antigen binding fragment thereof,and a pharmaceutically acceptable carrier. In another embodiment, theinvention provides a pharmaceutical composition comprising the antibody3G16 or an antigen binding fragment thereof, and a pharmaceuticallyacceptable carrier. In another embodiment, the invention provides apharmaceutical composition comprising the antibody 6L3 or an antigenbinding fragment thereof, and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical compositions of the invention maycomprise the above antibodies or antigen binding fragments thereof, asthe sole active ingredient. In another embodiment, the pharmaceuticalcomposition may comprise 2 or more, e.g., 2, 3, 4, 5, 6, 7, 8, or moreof the above antibodies or antigen binding fragment thereof. Asdiscussed herein, the pharmaceutical compositions of the invention mayalso comprise one or more antibodies, or antigen binding fragmentthereof, and a second antibody, or antigen binding fragment thereof,that neutralises hCMV infection.

Antibodies of the invention may be administered (either combined orseparately) with other therapeutics e.g. with chemotherapeuticcompounds, with radiotherapy, etc. Preferred therapeutic compoundsinclude anti-viral compounds such as ganciclovir, foscarnet andcidofovir. Such combination therapy provides an additive or synergisticimprovement in therapeutic efficacy relative to the individualtherapeutic agents when administered alone. The term “synergy” is usedto describe a combined effect of two or more active agents that isgreater than the sum of the individual effects of each respective activeagent. Thus, where the combined effect of two or more agents results in“synergistic inhibition” of an activity or process, it is intended thatthe inhibition of the activity or process is greater than the sum of theinhibitory effects of each respective active agent. The term“synergistic therapeutic effect” refers to a therapeutic effect observedwith a combination of two or more therapies wherein the therapeuticeffect (as measured by any of a number of parameters) is greater thanthe sum of the individual therapeutic effects observed with therespective individual therapies.

Antibodies may be administered to those patients who have previouslyshown no response to treatment for hCMV infection, i.e. have been shownto be refractive to anti-hCMV treatment. Such treatment may includeprevious treatment with an anti-viral agent. This may be due to, forexample, infection with an anti-viral resistant strain of hCMV.

In compositions of the invention that include antibodies of theinvention, the antibodies may make up at least 50% by weight (e.g. 60%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) of the totalprotein in the composition. The antibodies are thus in purified form.

The invention provides a method of preparing a pharmaceutical,comprising the steps of: (i) preparing an antibody of the invention; and(ii) admixing the purified antibody with one or morepharmaceutically-acceptable carriers.

The invention also provides a method of preparing a pharmaceutical,comprising the step of admixing an antibody with one or morepharmaceutically-acceptable carriers, wherein the antibody is amonoclonal antibody that was obtained from a transformed B cell of theinvention. Thus the procedures for first obtaining the monoclonalantibody and then preparing the pharmaceutical can be performed at verydifferent times by different people in different places (e.g. indifferent countries).

As an alternative to delivering antibodies or B cells for therapeuticpurposes, it is possible to deliver nucleic acid (typically DNA) thatencodes the monoclonal antibody (or active fragment thereof) of interestto a subject, such that the nucleic acid can be expressed in the subjectin situ to provide a desired therapeutic effect. Suitable gene therapyand nucleic acid delivery vectors are known in the art.

Compositions of the invention may be immunogenic compositions, and insome embodiments may be vaccine compositions comprising an antigencomprising an epitope in the hCMV UL128 protein, formed by the hCMVproteins UL130 and 131A, formed by the hCMV proteins UL128, UL130 andUL131A, formed by the hCMV proteins gH, gL, UL128 and UL130, in the hCMVgB protein, in the hCMV gH protein, or formed by the hCMV proteins gMand gN. Alternative compositions may comprise (i) an antigen comprisingan epitope formed by a combination of hCMV proteins UL128, UL130 andUL131A, and (ii) an antigen comprising an epitope found on gB, gH, gL,gM, gN, gO, UL128, UL130 or UL131A, or a combination thereof. Vaccinesaccording to the invention may either be prophylactic (i.e. to preventinfection) or therapeutic (i.e. to treat infection).

Compositions may include an antimicrobial, particularly if packaged in amultiple dose format. They may comprise a detergent e.g., a Tween(polysorbate), such as Tween 80. Detergents are generally present at lowlevels e.g. <0.01%. Compositions may also include sodium salts (e.g.sodium chloride) to give tonicity. A concentration of 10±2 mg/ml NaCl istypical.

Compositions may comprise a sugar alcohol (e.g. mannitol) or adisaccharide (e.g. sucrose or trehalose) e.g. at around 15-30 mg/ml(e.g. 25 mg/ml), particularly if they are to be lyophilised or if theyinclude material which has been reconstituted from lyophilised material.The pH of a composition for lyophilisation may be adjusted to around 6.1prior to lyophilisation.

The compositions of the invention may also comprise one or moreimmunoregulatory agents. In one embodiment, one or more of theimmunoregulatory agents include(s) an adjuvant.

The epitope compositions of the invention may elicit both a cellmediated immune response as well as a humoral immune response in orderto effectively address a hCMV infection. This immune response may inducelong lasting (e.g. neutralizing) antibodies and a cell mediated immunitythat can quickly respond upon exposure to hCMV.

Medical Treatments and Uses

The antibodies, antibody fragments of the invention or derivatives andvariants thereof may be used for the treatment of hCMV infection, forthe prevention of hCMV infection or for the diagnosis of hCMV infection.

Methods of diagnosis may include contacting an antibody or an antibodyfragment with a sample. Such samples may be tissue samples taken from,for example, salivary glands, lung, liver, pancreas, kidney, ear, eye,placenta, alimentary tract, heart, ovaries, pituitary, adrenals,thyroid, brain or skin. The methods of diagnosis may also include thedetection of an antigen/antibody complex.

The invention therefore provides (i) an antibody, an antibody fragment,or variants and derivatives thereof according to the invention, (ii) animmortalised B cell clone according to the invention, (iii) an epitopecapable of binding an antibody of the invention or (iv) a ligand,preferably an antibody, capable of binding an epitope that binds anantibody of the invention for use in therapy.

Also provided is a method of treating a patient comprising administeringto that patient (i) an antibody, an antibody fragment, or variants andderivatives thereof according to the invention, or, a ligand, preferablyan antibody, capable of binding an epitope that binds an antibody of theinvention.

The invention also provides the use of (i) an antibody, an antibodyfragment, or variants and derivatives thereof according to theinvention, (ii) an immortalised B cell clone according to the invention,(iii) an epitope capable of binding an antibody of the invention, or(iv) a ligand, preferably an antibody, that binds to an epitope capableof binding an antibody of the invention, in the manufacture of amedicament for the prevention or treatment of hCMV infection.

The invention provides a composition for use as a medicament for theprevention or treatment of an hCMV infection. It also provides the useof an antibody and/or a protein comprising an epitope to which such anantibody binds in the manufacture of a medicament for treatment of apatient and/or diagnosis in a patient. It also provides a method fortreating a subject in need of treatment, comprising the step ofadministering a composition of the invention to the subject. In someembodiments the subject may be a human. One way of checking efficacy oftherapeutic treatment involves monitoring disease symptoms afteradministration of the composition of the invention. Treatment can be asingle dose schedule or a multiple dose schedule.

In one embodiment, an antibody of the invention, an antigen-bindingfragment thereof, an epitope or a composition of the invention isadministered to a subject in need of such prophylactic or therapeutictreatment. Such a subject includes, but is not limited to, one who isparticularly at risk of, or susceptible to, hCMV infection. Exemplarysubjects include, but are not limited to, immunocompromised subjects orhCMV-seronegative or hCMV recently infected pregnant women. Exemplaryimmunocompromised subjects include, but are not limited to, thoseafflicted with HIV or those undergoing immunosuppressive therapy.

Antibodies of the invention and antigen-biding fragments thereof canalso be used in passive immunisation. Further, as described in thepresent invention, they may also be used in a kit for the diagnosis ofhCMV infection.

Epitopes capable of binding an antibody of the invention, e.g., themonoclonal antibodies 15D8, 4N10, 10F7, 10P3, 4122, 8L13, 2C12, 8C15,916, 7B13, 8J16, 8I21, 7I13, 7H3, 6B4, 5F1, 10C6, 4H9, 2B11, 11B12,13H11, 3G16, and 6L3, may be used in a kit for monitoring the efficacyof vaccination procedures by detecting the presence of protectiveanti-hCMV antibodies.

Antibodies, antibody fragment, or variants and derivatives thereof, asdescribed in the present invention may also be used in a kit formonitoring vaccine manufacture with the desired immunogenicity.

The invention also provides a method of preparing a pharmaceutical,comprising the step of admixing a monoclonal antibody with one or morepharmaceutically-acceptable carriers, wherein the monoclonal antibody isa monoclonal antibody that was obtained from an expression host of theinvention. Thus the procedures for first obtaining the monoclonalantibody (e.g. expressing it and/or purifying it) and then admixing itwith the pharmaceutical carrier(s) can be performed at very differenttimes by different people in different places (e.g. in differentcountries).

Starting with a transformed B cell of the invention, various steps ofculturing, sub-culturing, cloning, sub-cloning, sequencing, nucleic acidpreparation etc. can be performed in order to perpetuate the antibodyexpressed by the transformed B cell, with optional optimisation at eachstep. In a preferred embodiment, the above methods further comprisetechniques of optimisation (e.g. affinity maturation or optimisation)applied to the nucleic acids encoding the antibody. The inventionencompasses all cells, nucleic acids, vectors, sequences, antibodiesetc. used and prepared during such steps.

In all these methods, the nucleic acid used in the expression host maybe manipulated to insert, delete or amend certain nucleic acidsequences. Changes from such manipulation include, but are not limitedto, changes to introduce restriction sites, to amend codon usage, to addor optimise transcription and/or translation regulatory sequences, etc.It is also possible to change the nucleic acid to alter the encodedamino acids. For example, it may be useful to introduce one or more(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions,deletions and/or insertions into the antibody's amino acid sequence.Such point mutations can modify effector functions, antigen-bindingaffinity, post-translational modifications, immunogenicity, etc., canintroduce amino acids for the attachment of covalent groups (e.g.labels) or can introduce tags (e.g. for purification purposes).Mutations can be introduced in specific sites or can be introduced atrandom, followed by selection (e.g. molecular evolution). For instance,one or more nucleic acids encoding any of the CDR regions, heavy chainvariable regions or light chain variable regions of antibodies of theinvention can be randomly or directionally mutated to introducedifferent properties in the encoded amino acids. Such changes can be theresult of an iterative process wherein initial changes are retained andnew changes at other nucleotide positions are introduced. Moreover,changes achieved in independent steps may be combined. Differentproperties introduced into the encoded amino acids may include, but arenot limited to, enhanced affinity.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x means, for example,x+10%.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder” and“condition” (as in medical condition), in that all reflect an abnormalcondition of the human or animal body or of one of its parts thatimpairs normal functioning, is typically manifested by distinguishingsigns and symptoms, and causes the human or animal to have a reducedduration or quality of life.

As used herein, reference to “treatment” of a patient is intended toinclude prevention and prophylaxis. The term “patient” means all mammalsincluding humans. Examples of patients include humans, cows, dogs, cats,horses, goats, sheep, pigs, and rabbits. Generally, the patient is ahuman.

EXAMPLES

Exemplary embodiments of the present invention are provided in thefollowing examples. The following examples are presented only by way ofillustration and to assist one of ordinary skill in using the invention.The examples are not intended in any way to otherwise limit the scope ofthe invention.

Example 1: Cloning of B Cells and Screening for hCMV NeutralizingActivity

Donors with high hCMV neutralizing antibody titres in the serum wereidentified. Memory B cells were isolated and immortalised using EBV andCpG as described in reference 36. Briefly, memory B cells were isolatedby negative selection using CD22 beads, followed by removal of IgM⁻,IgD⁺ IgA⁺ B cells using specific antibodies and cell sorting. The sortedcells (IgG⁺) were immortalized with EBV in the presence of CpG 2006 andirradiated allogeneic mononuclear cells. Replicate cultures eachcontaining 50 memory B cells were set up in twenty 96 well U bottomplates. After two weeks the culture supernatants were collected andtested for their capacity to neutralize hCMV infection of eitherfibroblasts or epithelial cells in separate assays. B cell clones wereisolated from positive polyclonal cultures as described in reference 36.IgG concentrations in the supernatant of selected clones were determinedusing an IgG-specific ELISA.

For the viral neutralization assay a titrated amount of a clinical hCMVisolate was mixed with an equal volume of culture supernatant or withdilutions of human sera containing neutralizing antibodies. After 1 hourincubation at room temperature the mixture was added to confluentmonolayers of either endothelial cells (e.g. HUVEC cells or HMEC-1cells), epithelial cells (e.g. ARPE retinal cells), fibroblasts (e.g.MRC-9 or mesenchymal stromal cells) or myeloid cells (e.g.monocyte-derived dendritic cells) in 96 well flat-bottom plates andincubated at 37° C. for two days. The supernatant was discarded, thecells were fixed with cold methanol and stained with a mixture of mousemonoclonal antibodies to hCMV early antigens, followed by afluorescein-labeled goat anti mouse Ig. The plates were analyzed using afluorescence microscope. In the absence of neutralizing antibodies theinfected cells were 100-1,000/field, while in the presence of saturatingconcentrations of neutralizing antibodies the infection was completelyinhibited. The neutralizing titer is indicated as the concentration ofantibody (μg/ml) that gives a 50% or 90% reduction of hCMV infection.

Table 5A shows the neutralization of a hCMV clinical isolate (VR1814) onboth a fibroblastic cell line (MRC-9) and a human retinal epithelialcell line (ARPE). Some antibodies neutralized hCMV infection ofepithelial cells (ARPE) but they did not neutralize infection offibroblasts (MRC-9). This agrees with previous data that differentproteins are responsible for tropism towards a particular cell type [7].Most of these antibodies, which are specific for one or more proteins ofthe gH/gL/UL128/UL130/UL131A protein complex, neutralized hCMV infectionof epithelial cells at very low concentrations (50% reduction of hCMVinfection at concentrations ranging from 0.01 μg/ml and 0.001 μg/ml).Other antibodies, which are specific for the hCMV protein gB, gH or acombination of gM and gN, neutralized hCMV infection of fibroblasts andepithelial cells with comparable potency. These results show that someof the hCMV neutralizing antibodies are equally potent on bothfibroblasts and epithelial cells, while others show differentialactivity on the two cell types.

Based on the analysis shown in Table 5A, antibodies were grouped intoGroup 1 (neutralizing hCMV infection of both fibroblasts and epithelialcells) and Group 2 (neutralizing hCMV infection of epithelial cells).Table 5B shows an independent experiment performed using purifiedantibodies. The results show that Group 2 antibodies neutralizedinfection of epithelial cells with IC90 values (i.e. the concentrationof antibody required to give 90% reduction of viral infection) rangingfrom 0.007 μg/ml to 0.003 μg/ml while Group 1 antibodies neutralizedinfection of both fibroblasts and epithelial cells with IC90 valuesranging from 0.1 μg/ml to 30 μg/ml. Group 2 antibodies also neutralizedinfection of endothelial cells (HUVEC) and myeloid cells(monocyte-derived dendritic cells) (data not shown). Group 1 antibodiesalso neutralized infection of endothelial cells (HUVEC), myeloid cells(monocyte-derived dendritic cells) and bone marrow mesenchymal stromalcells, as shown for some representative antibodies in Table 5C.Antibodies of the invention also neutralized infection of endothelialcells (HUVEC) by different hCMV clinical isolates: VR6952 (from urine),VR3480B1 (from blood, ganciclovir-resistant) and VR4760 (from blood,ganciclovir and foscarnet-resistant) (data not shown).

It is anticipated that antibodies that neutralize infection of differentcell types may be combined to bring about an additive or synergisticneutralization effect when the different cell types are present duringinfection. As one example, a neutralizing antibody, such as 15D8 whichis potent in neutralizing infection of epithelial cells but does notneutralize infection of fibroblasts might be combined with 3G16 whichdoes have virus neutralizing activity on fibroblasts. As anotherexample, a neutralizing antibody, such as 916 which is potent inneutralizing infection of epithelial cells but does not neutralizeinfection of fibroblasts, might be combined with 6B4 which does havevirus neutralizing activity on fibroblasts.

TABLE 5A 50% 50% Neutrali- Neutrali- zation⁽¹⁾ zation⁽¹⁾ mAb DonorSpecificity⁽²⁾ MRC-9 ARPE 15D8 GRA UL128 − ++++ 4N10 GIO UL130/UL131A +++++ 10F7 PAP UL130/UL131A + +++ 10P3 PEL UL130/UL131A − ++++ 4I22 PELUL130/UL131A − +++ 8L13 PEL UL130/UL131A − +++ 2C12 PAPUL128/UL130/UL131A + +++ 7B13 PAP UL128/UL130/UL131A − ++++ 7I13 PAPUL128/UL130/UL131A − +++ 8C15 PAP UL128/UL130/UL131A − ++++ 8J16 PAPUL128/UL130/UL131A − ++++ 9I6 PEL UL128/UL130/UL131A − ++++ 8I21 PELgH/gL/UL128/UL130 − +++ 11B12 PAP gH + + 13H11 GRA gH + +++ 3G16 PELgH + + 7H3 PEL gB + − 10C6 PEL gB + + 5F1 PEL gB + + 6B4 PEL gB + + 4H9PEL gB + + 6L3 PEL gM/gN Not done + ⁽¹⁾Values indicating theconcentration of antibody required to give a 50% reduction of hCMVinfection of fibroblasts (e.g. MRC-9) or epithelial cells (e.g. ARPEretinal cells). Concentration as follows: ++++ < 0.001 μg/ml; +++ < 0.01μg/ml; ++ < 0.1 μg/ml; + ≦ 2 μg/ml; − Not neutralizing at the highestconcentration tested (2 μg/ml). ⁽²⁾Specificity as defined in Table 6.

TABLE 5B 90% 90% Neutrali- Neutrali- zation⁽¹⁾ zation⁽¹⁾ Group mAb DonorSpecificity⁽²⁾ MRC-9 ARPE 2 15D8 GRA UL128  nn⁽³⁾ 0.008 2 4N10 GIOUL130/UL131A nn 0.02 2 10F7 PAP UL130/UL131A nn 0.002 2 10P3 PELUL130/UL131A nn 0.0025 2 4I22 PEL UL130/UL131A nn 0.0015 2 8L13 PELUL130/UL131A nn 0.001 2 2C12 PAP UL128/UL130/UL131A nn 0.006 2 7B13 PAPUL128/UL130/UL131A nn 0.003 2 7I13 PAP UL128/UL130/UL131A nn 0.008 28C15 PAP UL128/UL130/UL131A nn 0.0025 2 8J16 PAP UL128/UL130/UL131A nn0.0008 2 9I6 PEL UL128/UL130/UL131A nn 0.0007 2 8I21 PELgH/gL/UL128/UL130 nn 0.03 1 11B12 PAP gH 3.5 1.2 1 13H11 GRA gH 1.12 0.41 3G16 PEL gH 1.0 0.3 1 7H3 PEL gB 3 0.6 1 10C6 PEL gB 0.75 0.2 1 5F1PEL gB 0.5 0.1 1 6B4 PEL gB 1.0 0.15 1 4H9 PEL gB 10 0.4 1 2B11 PEL gB0.75 0.2 1 6L3 PEL gM/gN 30 10 ⁽¹⁾Values indicating the concentration ofantibody in μg/ml required to give a 90% reduction of hCMV (VR1814)infection of fibroblasts (e.g. MRC-9) or epithelial cells (e.g. ARPEretinal cells). ⁽²⁾Specificity as defined in Table 6. ⁽³⁾nn, notneutralizing at the highest concentration tested (10 μg/ml).

TABLE 5C 50% Neutralization⁽¹⁾ Group mAb Specificity HUVEC Mo-DC BM-MSC1 7H3 gH nd 0.06 2 1 10C6 gH 0.19 0.02 0.3 1 5F1 gH 0.21 0.05 0.3 1 6B4gH nd 0.11 2 ⁽¹⁾Values indicating the concentration of antibody in μg/mlrequired to give a 50% reduction of hCMV (VR1814) infection of primarycells. HUVEC, human umbilical vein endothelial cells, Mo-DC,monocyte-derived dendritic cells, BM-MSC, mesenchymal bone-marrowstromal cells.

Example 2: Identification of the Target Antigens Recognized By theMonoclonal Antibodies

To map the specificity of the hCMV neutralizing antibodies, HEK293Tcells were transfected with one or more vectors encoding full lengthhCMV proteins UL128, UL130, UL131A, gH, gL, gB, gM, and gN. After 36 h,cells were fixed, permeabilized and stained with the human monoclonalantibodies followed by goat anti-human IgG. FIG. 1 shows the binding ofrepresentative antibodies to HEK293T cells expressing one or more hCMVproteins. Table 6 shows the staining pattern of all the differentantibodies to hCMV gene-transfected HEK293T cells. With the exception ofantibody 15D8, that stained UL128-transfected cells, all the other Group2 antibodies did not stain single gene transfectants, suggesting thatthey may recognize epitopes that require co-expression of more than onegene product. Indeed, five antibodies (4N10, 10F7, 10P3, 4I22 and 8L13)stained cells co-expressing UL130 and UL131A, six antibodies (2C12,7B13, 7I13, 8C15, 8J16 and 9I6) stained cells co-expressing UL128, UL130and UL131A, and one antibody (8I21) stained cells transfected with UL128and UL130 as well as with gH and gL. All these antibodies also stainedHEK293T cells transfected with all genes forming the gH/gL/UL128-130complex. Among the Group 1 antibodies, three (11B12, 13H11 and 3G16)stained cells expressing the hCMV protein gH, six (7H3, 10C6, 5F1, 6B4,4H9 and 2B11) stained cells expressing the hCMV protein gB and one (6L3)stained cells coexpressing the hCMV proteins gM and gN.

TABLE 6 Monoclonal antibody Group 2 Group 1 2C12 7H3 4N10 7B13 10C6 10F77I13 5F1 10P3 8C15 11B12 6B4 HEK293T cells transfected 4I22 8J16 13H114H9 with: 15D8 8L13 9I6 8I21 3G16 2B11 6L3 UL128 + − − − − − nd⁽¹⁾ UL130− − − − − − nd UL131A − − − − − − nd UL128 + UL130 + − − − − − ndUL128 + UL131A + − − − − − nd UL130 + UL131A − + − − − − nd UL128 +UL130 + UL131A + + + − − − − gH − − − − + − − gH + gL − − − − + − − gH +UL128 + UL130 + UL131A + + + − + nd nd gL + UL128 + UL130 + UL131A + + +− − nd nd gH + gL + UL128 + − − − + nd nd gH + gL + UL130 − − − − + ndnd gH + gL + UL131A − − − − + nd nd gH + gL + UL128 + UL130 + − − + + ndnd gH + gL + UL128 + UL130 + UL131A + + + + + − − gB − − − nd − + − gMnd − − nd nd nd − gN nd − − nd nd nd − gM + gN − − − − nd nd + ⁽¹⁾nd,not done.

To further explore the identity of the antigen sites to which theantibodies bind, cross-competition experiments were performed. Here,HEK293T cells were transfected with vectors encoding full length hCMVproteins gH, gL, UL128, UL130 and UL131A. The cells were then incubatedwith a 20-fold excess of a competitor hCMV neutralizing antibody beforeaddition of a biotinylated antibody. This procedure was repeated severaltimes with different competitor antibodies and biotinylated antibodies.In these experiments four antibodies described in patent applicationSer. No. 11/969,104 (11F11, 2F4 and 5A2) and patent application Ser. No.12/174,568 (6G4) were included. The data is shown in Table 7A, B.

TABLE 7A Competitor Inhibition of binding (%) (20-fold 15D8- 4N10- 10F7-4I22- 1F11- 2F4- 5A2- excess) Specificity⁽¹⁾ biotin biotin biotin biotinbiotin biotin biotin 15D8 UL128 100  0 0 0 0 0 0 4N10 UL130/UL131A 0 1000 0 0 0 100 10F7 UL130/UL131A 0 0 100 100 100 100  0 10P3 UL130/UL131A 0nd nd 0 0 0 Nd 4I22 UL130/UL131A nd 0 100 100 100 100  0 8L13UL130/UL131A nd nd 100 nd 100 Nd nd 1F11 UL130/UL131A 0 0 100 100 100100  0 2F4 UL130/UL131A nd 0 100 100 100 100  0 5A2 UL130/UL131A nd 1000 0 0   50⁽²⁾ 100 2C12 UL128/UL130/UL131A 0 0 0 0 0 0 0 7B13UL128/UL130/UL131A nd nd nd nd nd nd nd 7I13 UL128/UL130/UL131A nd nd ndnd 0 nd nd 8C15 UL128/UL130/UL131A nd nd nd 0 nd nd nd 8J16UL128/UL130/UL131A nd nd nd 0 0 0 nd 9I6 UL128/UL130/UL131A nd nd Nd 0 00 nd 6G4 UL128/UL130/UL131A 0 0 0 0 0 0 0 8I21 gH/gL/UL128/UL130 0 90 nd0 0 0 95 ⁽¹⁾Specificity as defined is Table 6. ⁽²⁾Competition below 100%may be due to partial overlap of epitopes or to steric hindrance or tolower affinity.

TABLE 7B Competitor Inhibition of binding (%) (20-fold 2C12- 8C15- 8J16-9I6- 6G4- 8I21- excess) Specificity⁽¹⁾ biotin biotin biotin biotinbiotin biotin 15D8 UL128 0 nd nd nd 0 0 4N10 UL130/UL131A 0 nd nd nd 0  90⁽²⁾ 10F7 UL130/UL131A 0 nd nd nd 0 0 10P3 UL130/UL131A 0 nd nd nd 00 4I22 UL130/UL131A 0 nd 0 nd nd 0 8L13 UL130/UL131A nd nd nd nd nd nd1F11 UL130/UL131A 0 nd nd nd 0 0 2F4 UL130/UL131A 0 nd nd 0 0 0 5A2UL130/UL131A 0 nd nd 0 0 92  2C12 UL128/UL130/UL131A 100 100 100 100 1000 7B13 UL128/UL130/UL131A 100 100 100 100 100 0 7I13 UL128/UL130/UL131A0 0 0 0 0 0 8C15 UL128/UL130/UL131A 100 100 100 100 100 0 8J16UL128/UL130/UL131A 100 100 100 70 100 0 9I6 UL128/UL130/UL131A 100 100100 100 100 0 6G4 UL128/UL130/UL131A 100 100 100 100 100 0 8I21gH/gL/UL128/UL130 0 nd nd nd 0 100  3G16 gH 0 nd nd nd 0 0⁽¹⁾Specificity as defined is Table 6. ⁽²⁾Competition below 100% may bedue to partial overlap of epitopes or to steric hindrance or to loweraffinity.

Based on the data in Table 7A, B, at least seven distinct antigenicsites can be distinguished on the hCMV complex formed by gH, gL, UL128and UL130 (Table 8). Site 1 is present in UL128 and is defined byantibody 15D8. Sites 2 to 4 are formed by the combination of UL130 andUL131A and are defined by the antibodies 10F7 4122, 8L13, 1F11 and 2F4(site 2), by 4N10 and 5A2 (site 3), and by 10P3 (site 4), respectively.Sites 5 and 6 are formed by the combination of UL128, UL130 and UL131Aand are defined by antibodies 2C12, 7B13, 8C15, 8J16, 916 and 6G4 (site5) and by 7I13 (site 6), respectively. Finally, site 7 is formed by thecombination of gH, gL, UL128 and UL130 and is defined by the antibody8I21. Antibodies defining site 7 and site 3 partially competed with eachother, suggesting that these sites may be close in the structure of thegH/gL/UL128-131A complex.

It is anticipated that neutralizing antibodies targeted to differentepitopes on the same target can be used in combination to achieve robustneutralization of virus infection, as exemplified by 10F7 and 4N10 or by8J16 and 7I13. Moreover, it is anticipated that neutralizing antibodiestargeted to different target molecules or combinations of targetmolecules may be used together to achieve robust virus neutralization.As one example, Table 8 suggests that 15D8 and 10F7, 15D8 and 2C12, or8J16 and 8I21 could be combined to bring about additive or synergenichCMV neutralization effects.

TABLE 8 Antigenic Antibodies defining Target antigen site the antigenicsite UL128 1 15D8 UL130/UL131A 2 10F7, 4I22, 8L13, 1F11, 2F4UL130/UL131A 3 4N10, 5A2 UL130/UL131A 4 10P3 UL128/UL130/UL131A 5 2C12,7B13, 8C15, 8J16, 916, 6G4 UL128/UL130/UL131A 6 7I13 gH/gL/UL128/UL130 78I21

In a manner similar to what described in Table 7, HEK293T cells weretransfected with a vector encoding full length gH to examine thecross-competition binding of the anti-gH antibodies. As can be seen inFIG. 2A and Table 9, at least two different binding sites wereidentified in the hCMV gH protein. The antibody 3G16 defines one siteand the antibodies 11B12 and 13H11 define a second site. Finally,HEK293T cells were transfected with a vector encoding full length gB toexamine the cross-competition binding of the anti-gB antibodies. As canbe seen in FIG. 2B and Table 10, at least three different antigenicsites were identified in the hCMV gB protein. The antibody 6B4 definesone site, 7H3 defines a second site and the set of 10C6, 5F1, 4H9 and2B11 define a third site. Antibody 6B4 (recognizing gB site 1) reactedby ELISA with the gB 69-78 peptide (EC₅₀ of 0.044 μg/ml). It isanticipated that antibodies that target different sites even on the sametarget molecule can be used in combination to achieve robust virusneutralization. It is anticipated that antibodies that target differentsites even on the same target molecule can be used in combination toachieve robust virus neutralization.

TABLE 9 Inhibition of binding (%) of: Competitor 3G16- 11B12- 13H11-Antigenic 20-fold excess Specificity⁽¹⁾ biotin biotin biotin site in gH3G16 gH 100 0 0 1 11B12 gH 0 100 100 2 13H11 gH 0 100 100 2 ⁽¹⁾Asdefined in Table 6.

TABLE 10 Inhibition of binding (%) of: Competitor 7H3- 10C6- 5F1- 6B4-4H9- 2B11- Antigenic 20-fold excess Specificity⁽¹⁾ biotin biotin biotinbiotin biotin biotin site in gB 6B4 gB 0 0 0 100 0 0 1 7H3 gB 100 0 0 00 0 2 10C6 gB 0 100 100 0 100 100 3 5F1 gB 0 100 100 0 100 100 3 4H9 gB0 100 100 0 100 100 3 2B11 gB 0 100 100 0 100 100 3 ⁽¹⁾As defined inTable 6. 2) Competition below 100% may be due to partial overlap ofepitopes, to steric hindrance or to lower affinity.

To summarize, 15D8 binds to an epitope in UL128 that is distinct fromthe epitope recognized by 2C12, 7B13, 6G4 (all specific for acombination of UL128, UL130 and UL131A) and from the epitope recognizedby 8I21 (specific for a combination of gH, gL, UL128 and UL130). Inaddition binding of 15D8 to its epitope is not inhibited by 4N10, 10F7,10P3 and 1F11 (all specific for a combination of UL130 and UL131A).

4N10 binds to an epitope which requires expression of UL130 and UL131Aand that is the same or largely overlapping to the epitopes recognizedby 5A2 (specific for a combination of UL130 and UL131A) and 8I21(specific for a combination of gH, gL, UL128 and UL130) but distinctfrom the epitopes recognized by 10F7, 4I22, 1F11, 2F4 (all specific fora combination of UL130 and UL131A), 2C12 and 6G4 (both specific for acombination of UL128, UL130 and UL131A). In addition binding of 4N10 toits epitope is not inhibited by 15D8 (specific for UL128).

10F7 binds to an epitope which requires expression of UL130 and UL131Athat is the same or largely overlapping to the epitope(s) recognized by4I22, 8L13, 1F11 and 2F4 but distinct from epitope(s) recognized by 4N10and 5A2 (both specific for a combination of UL130 and UL131A) as well asdistinct from epitopes recognized by 2C12 and 6G4 (both specific for acombination of UL128, UL130 and UL131A). In addition binding of 10F7 toits epitope is not inhibited by 15D8 (specific for UL128) or by 13H11(specific for gH).

4I22 binds to an epitope which requires expression of UL130 and UL131Aand that is the same or partially overlapping to epitope(s) recognizedby 2F4, 1F11 and 10F7 but distinct from epitope(s) recognized by 4N10,10P3 and 5A2 (all specific for a combination of UL130 and UL131A) aswell as distinct from the epitopes recognized by 2C12, 8C15, 8J16, 916,6G4 (all specific for a combination of UL128, UL130 and UL131A) and 8I21(specific for a combination of gH, gL, UL128 and UL130. In additionbinding of 4I22 to its epitope is not inhibited by the antibodies 15D8(specific for UL128) or by 13H11 (specific for gH).

2C12 binds to an epitope which requires expression of hCMV UL128, UL130and UL131A gene products and that is the same or largely overlapping toepitope(s) recognized by 7B13, 8C15, 8J16, 9I6 and 6G4 but distinct fromthe epitope recognized by 7I13 (all specific for a combination of UL128,UL130 and UL131A) and distinct from epitope(s) recognized by 15D8(specific for UL128), 4N10, 10F7, 10P3, 4122, 8L13, 1F11, 2F4, 5A2 (allspecific for a combination of UL130 and UL131A) and 8I21 (specific for acombination of gH, gL, UL128 and UL130). In addition binding of 2C12 toits epitope is not inhibited by 3G16 (specific for gH).

8C15 binds to an epitope which requires expression of hCMV UL128, UL130and UL131A gene products and that is the same or largely overlapping toepitope(s) recognized by 2C12, 7B13, 8J16, 9I6 and 6G4 but distinct fromthe epitope recognized by 7I13 (all specific for a combination of UL128,UL130 and UL131A).

8J16 binds to an epitope which requires expression of hCMV UL128, UL130and UL131A gene products and that is the same or largely overlapping toepitope(s) recognized by 2C12, 7B13, 8C15, 916 and 6G4, but distinctfrom the epitope recognized by 7I13 (all specific for a combination ofUL128, UL130 and UL131A) and from the epitope recognized by 4I22(specific for a combination of UL130 and UL131A).

9I6 binds to an epitope which requires expression of hCMV UL128, UL130and UL131A gene products and that is the same or largely overlapping toepitope(s) recognized by 2C12, 7B13, 8C15, 8J16 and 6G4 but distinctfrom the epitope recognized by 7I13 (all specific for a combination ofUL128, UL130 and UL131A) and from the epitope(s) recognized by 2F4 and5A2 (specific for a combination of UL130 and UL131A).

8I21 binds to an epitope which requires expression of hCMV gH, gL, UL128and UL130 gene products and that may be partially overlapping toepitope(s) recognized by 4N10 and 5A2 (both specific for a combinationof UL130 and UL131A) but distinct from epitopes recognized by 15D8(specific UL128), 10F7, 10P3, 4122, 1F11, 2F4 (all specific for acombination of UL130 and UL131A), 2C12, 7B13, 7I13, 8C15, 8J16, 916 and6G4 (all specific for a combination of UL128, UL130 and UL131A). Inaddition binding of 8I21 to its epitope is not inhibited by 3G16(specific for gH).

3G16 binds to an epitope in gH that is distinct from the epitope(s)recognized by 11B12 and 13H11 (both specific for gH).

11B12 binds to an epitope in gH that is the same or largely overlappingto the epitope recognized by 13H11 and distinct from the epitopesrecognized by 3G16 (both specific for gH).

13H11 binds to an epitope in gH that is the same or largely overlappingto the epitope recognized by 11B12 and distinct from the epitopesrecognized by 3G16 (both specific for gH).

6B4 recognizes an epitope in gB that is distinct from the epitope(s)recognized by 7H3, 4H9, 5F1, 10C6 and 2B11 (all specific for gB).

7H3 binds to an epitope in gB that is distinct from the epitope(s)recognized by 6B4, 7H3, 4H9, 5F1, 10C6 and 2B11 (all specific for gB).

10C6 binds to an epitope in gB that is the same or partially overlappingto the epitope(s) recognized by 5F1, 4H9 and 2B11, but distinct from theepitope(s) recognized by 7H3 and 6B4 (all specific for gB).

5F1 binds to an epitope in gB that is the same or largely overlapping tothe epitope(s) recognized by 10C6, 4H9 and 2B11 but distinct from theepitope(s) recognized by 6B4 and 7H3 (all specific for gH).

4H9 binds to an epitope in gB that is the same or largely overlapping tothe epitope(s) recognized by 5F1, 10C6 and 2B11, but distinct from theepitope(s) recognized by 6B4 and 7H3 (all specific for gH).

2B11 binds to an epitope in gB that is the same or largely overlappingto the epitope(s) recognized by 5F1, 10C6 and 4H9 but distinct from theepitope(s) recognized by 6B4 and 7H3 (all specific for gH).

Example 3: Breadth of Neutralizing Activity of Antibody 15D8

UL128 is the most conserved gene of the UL132-128 locus. However,sequences derived from several clinical isolates revealed the existenceof 10 variants with one or more mutations when compared to the VR1814sequence. We therefore investigated whether the binding of theUL128-specific antibody 15D8 would be affected by any of thesemutations. To this aim, published amino acid sequences of variants ofUL128 from clinical isolates (VR4603-M, VR4836-M, VR5001-M, VR4254-M,VR4969-M, VR4313-M, VR4116-M, VR5235-T, VR5055-T, VR4168-A, VR1814-PCR)and laboratory strains (Towne, TB40/E, AD169, Merlin and Toledo) werealigned, and a gene was synthesized encoding a protein that includes allamino acid substitutions described as well as an additional mutationthat we found to be generated at very high frequency in vitro upon PCRamplification (F33V). The nucleotide sequence of the synthetic gene was:

(SEQ ID NO: 371) atgaacagcaaagacctgacgccgttcttgacgaccttgtggctgctattggaccacagccgcgtgccgcgggtacgcgcagaagaatgttgcgaattcataaacgtcaaccacccgccggaacgctgttacgatttcaaaatgtgcaatctgttcaccgtcgcgctgcggtgtccggacggcgaagtctgctacagtcccgagaaaacggctgagattcgcgggatcgtcaccaccatgacccattcattgacacgccaggtcatccacaacaaactgacgagctgcaactacaatccgttatacctcgaagctgacgggcgaatacgctgcggcaaagtgagcgacaaggcgcagtacctgctgggcgccgctggcagcgttccctatcgatggatcaacctggaatacgacaagataacccggatcgtgggcctggatcagtacctggagagcgttaagaaacacaaacggctggatgtgtgccgcgctaaaatggg ctatatgctgcagtag.

HEK293T cells were transfected with the original UL128 from VR1814 orwith the pan-mutated gene and stained with serial dilutions of 15D8antibody. As shown in FIG. 3, the original and the pan-mutated UL128protein were recognized by 15D8 with comparable efficiency (saturatedstaining at ˜0.2 μg/ml). These findings indicate that 15D8 recognize ahighly conserved epitope in the UL128 encoded protein.

All patents and publications referred to herein are expresslyincorporated by reference in their entirety.

It should be noted that there are alternative ways of implementing thepresent invention and that various modifications can be made withoutdeparting from the scope and spirit of the invention. Accordingly, thepresent embodiments are to be considered as illustrative and notrestrictive, and the invention is not to be limited to the details givenherein, but may be modified within the scope and equivalents of theappended claims.

REFERENCES (THE CONTENTS OF WHICH ARE HEREBY INCORPORATED BY REFERENCE)

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The invention claimed is:
 1. A composition comprising an isolatedantibody, or an antigen binding fragment thereof, comprising the CDR1,CDR2, and CDR3 sequences of the heavy and light chain variable regionsequences set forth in SEQ ID NOs: 228 and 229, respectively, whereinthe composition is in lyophilized form.
 2. A composition comprising anisolated antibody, or an antigen binding fragment thereof, comprisingheavy chain variable region CDR1, CDR2, and CDR3 sequences set forth inSEQ ID NOs: 216, 217, and 218, respectively, and light chain variableregion CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 219, 220,and 221, respectively wherein the composition is in lyophilized form. 3.The composition of claim 2, wherein the antibody, or antigen bindingfragment thereof, comprises the heavy and light chain variable regionsequences set forth in SEQ ID NOs: 228 and 229, respectively.
 4. Thecomposition of claim 1, wherein the antibody or fragment is specific fora human cytomegalovirus (hCMV) protein gH.
 5. The composition of claim1, further comprising an adjuvant or a sufficient amount of apreservative.
 6. The composition of claim 1, wherein the antibody orfragment inhibits infection of epithelial cells, wherein theconcentration of antibody required for 50% inhibition of hCMV is 0.01μg/ml or less.
 7. The composition of claim 1, wherein the antibody orfragment is a human antibody, a monoclonal antibody, a single chainantibody, Fab, Fab′, F(ab′)2, Fv or scFv.
 8. The composition of claim 7,further comprising at least one additional antibody, or an antigenbinding fragment thereof, which inhibits hCMV infection.
 9. Thecomposition of claim 8, wherein the additional antibody binds to an hCMVgB protein, an hCMV UL128 protein, an hCMV gH protein, a complex formedby hCMV UL130/UL131A proteins, a complex formed by hCMVUL128/UL130/UL131A proteins, a complex formed by hCMV gH/gL/UL128/UL130proteins, a complex formed by hCMV gM/gN proteins, or a combinationthereof.
 10. A method of inhibiting hCMV infection in a subject,comprising administering an effective amount of the composition of claim1, wherein the composition is reconstituted prior to injection orinfusion and wherein hCMV infection is inhibited.
 11. The method ofclaim 10, further comprising the step of administering a secondcomposition comprising a second antibody, or an antigen binding fragmentthereof, which inhibits hCMV infection, wherein the compositions areadministered simultaneously or sequentially.
 12. A method of inhibitinghCMV infection in an isolated cell, comprising contacting the cell withan effective amount of the composition of claim 1, followingreconstitution of the composition, wherein hCMV infection is inhibited.13. The method of claim 12, further comprising the step of contactingthe cell with a second composition comprising a second antibody, or anantigen binding fragment thereof, which inhibits hCMV infection, whereinthe cell is contacted by the compositions simultaneously orsequentially.
 14. A kit comprising the composition of claim 1 and apackage insert comprising instructions for administering of thecomposition for inhibiting hCMV infection.
 15. The kit of claim 14,wherein the antibody or fragment comprises the heavy and light chainvariable region sequences set forth in SEQ ID NOs: 228 and 229,respectively.
 16. The kit of claim 14, further comprising at least oneadditional antibody, or an antigen binding fragment thereof, whichinhibits hCMV infection.
 17. The kit of claim 16, wherein the additionalantibody binds to an hCMV gB protein, an hCMV UL128 protein, an hCMV gHprotein, a complex formed by hCMV UL130/UL131A proteins, a complexformed by hCMV UL128/UL130/UL131A proteins, a complex formed by hCMVgH/gL/UL128/UL130 proteins, a complex formed by hCMV gM/gN proteins, ora combination thereof.